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The neurosecretory cells of barnacles
J. exp. mar. Biol. Ecol., 1967, Vol.
1, pp. 154-l 69; North-Holland
THE NEUROSECRETORY
Publishing Company,
Amsterdam
CELLS OF BARNACLES
D. B. MCGREGOR 1 Departmentof Zoofogy,King’s College, London, Ettglmd Abstract: The central nervous systems of several species of acorn barnacles have been examined for the presence of so-called neurosecretory granules. PAF-staining granules were found in all the species but only in Bulunus perfbratus could CHP-staining granules be found. In this species also, colloidal accumulations were found which were reminiscent of the refractive material which is so characteristic of the sinus glands and other neurosecretory centres in malacostracans. The granules also stained with Sudan black, when dissolved in propylene glycol, and 1~x01 fast blue, but the use of stains for proteins was unsuccessful. This is rather different from the results obtained with ne~rosecretory granules from a variety of other sources. The staining characteristics of the granules from these acorn barnacles were also slightly different from those reported for the stalked barnacle, Pollicipespolymerus. A long-term cycle of PAF-staining granule concentrations, although looked for in Balanus baianoides, could not be found. It is concluded that there is evidence suggestive of neurosecretory activity but that neither the histological nor the physiological approaches have, as yet, provided unequivocal answers.
INTRODUCTION
Apart from the decapods, the crustaceans have received scant attention from physiologists engaged on neurosecretory studies. Within the last ten years, however, several attempts have been made to apply the methods so suc~ssfully used with decapods to the entomostracans. Some success has been achieved, particularly with the cirripedes. In this paper the results are reported of a histological study on the nerve cells of various barnacles, and their bearing upon the problem of neurosecretion in these animals is discussed. MATERIALS AND METHODS
Most of this work has been done on Balanus balanoides and B. perforatus; in addition B. crenatus, B. humeri, B. spongicola, Chthamalus stellatus, Verruca stroemia, and Elminius modestus have been investigated. If the central nervous system (CNS) were to be examined fresh, the soft part of the animal was removed from its shell and the dissection carried out with the live animal immersed in sea water. The animal was held in its normal position by one pin through the anterior part of the prosoma and another bent over the cirri. By carefully removing the mouth cone, the CNS, which lies directly beneath it, could be seen in a bright light. The long nerves from the s~boesophageal ganglion leading to the cirri and to 1 Present address: Flour-Millers’
and Bakers’
Research
England 154
Association,
Old London Road, St. Album,
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155
the prosoma as well as those from the tiny supraoesophogeal ganglion were cut and the principal ganglia eased away from the surrounding tissue. When the material was to be examined histologically, the soft parts were removed from the shell and fixed in alcoholic Bouin’s fluid or in neutral buffered formalin. Following dehydration in cellosolve, the specimens were cleared in benzene and then infiltrated and embedded in paraffin wax (m.p. 52 “C). Sections were cut at 7~, smoothed out by floating on warm water and allowed to dry on clean glass slides. No affixative was used. The staining techniques used were as follows:
(i) Periodic acid-Schiff (PAS) for glycolic groups (Pearse, 1961, after ~otchkiss). (ii) Amylase-PAS for distinguishing glycogen from other glycol-containing substances. (iii) Amylase-paraldehyde fuchsin to remove glycogen before staining neurosecretory substances. (iv) Alcian blue for acid mucopolysaccharides (Steedman, 1950). Lipids
(i) Sudan black B in propylene glycol for neutral fats and lipoproteins (Chiffelle & Putt, 1951). (ii) Luxoi fast blue (a copper phthalocyanin~ for phospholipids and lipoproteins (Kluver & Barrera, 1953). Proteins
(i) Ninhydrin-Schiff for protein bound -NH, groups (Yasuma & Ichikawa, 1953). (ii) Performic acid - Victoria blue (PFAVB) for sulphydryl groups (Humberstone, personal communication). This is a semi-specific stain for neurosecretory substances. (iii) Mercury-bromophenol blue for proteins (Bonhag, 1955). (iv) Pepsin-paraldehyde fuchsin. Incubation as recommended by Pearse (1961). (v) Trypsin-paraldehyde fuchsin. Incubation as recommended by Pearse (1961).
(i) Chrome haematoxylin-phlo~ne B (CHP) ~Bargmann, 1949). This is a semispecific stain for neurosecretory substances. (ii) Paraldehyde fuchsin (PAF) (Gabe, 1953). Used with preliminary oxidation as in the CHP technique, this is a semi-specific stain for neurosecretory material. (iii) Acid-permanganate-Schiff for the demonstration of aldehydes exposed by the oxidation procedures used for CHP and PAF staining. (iv) Acid-permanganate-diedone-PAF to block aldehydes produced in the oxidation procedure. Blocking with dimedone as recommended by Pearse (1961). (v) Acid-permanganate-dimedone-Schiff. Used to check that the blockage of aldehydes by dimedone had been successful.
156
D. B. MCGREGOR
(vi) Schmorl’s ferricyanide for lipofuscin, melanin, and argentaffin granules (Pearse, 1961). (vii) Long-Ziehl-Ne~ls~n method for acid-fast lipofuscins and lipoproteins (Pearse, 1961). In order to investigate the possibility of a long term cycle of secretory activity, Balunus balunoides was sampled at one to two weekly intervals from both Brighton and Bradwell-on-Sea between April 1964 and August 1965. From each sample random sections through the CNS of about six individuals were stained and examined. RESULTS
The general histology of the CNS has been described (Barnes & Gonor, 1958a). The outstanding feature of the neurones is the numerous vacuoles of varying sizes which are always present peripherally in the cell-bodies and which may fill them to such an extent that the cytoplasm is reduced to a few thin strands surrounding the vacuoles and the cell nucleus. These vacuoles may be partly fixation artefacts but large vacuoles are also visible in nerve cells teased from fresh nervous tissues of B. balanoides and their contents can be seen oozing out of the cell when squashed. Nevertheless, the shock caused by the dissection procedure may be enough to produce great morphological changes in the cytoplasm. As with the species described by Barnes & Gonor, the present species had two kinds of cell in their ganglia. The cells of B. perfuratus will be described in detail but those of the other species examined are similarly differentiated into two types. Cell dimensions are di~cult to determine accurately because the vacuoles in the cytoplasm distort the cell outline and often make it impossible to distinguish between the cytoplasm of one cell and that of an adjacent one. The nuclei of the small cell type average 4.2 p in diameter and are found in all parts of the ganglia where cell bodies occur. In the fused suboesophageal ganglion these small nuclei are found mainly on the dorsal side although they are also present in the ventral, lateral and central parts of the ganglion. The cell bodies of these small neurones average 9.5 I( diameter; hence there is very little cytoplasm in the cell bodies and the structured cytoplasm (in sectioned material) may be further reduced by the presence of vacuoles. No basophilic Nissl zone was ever observed. The nucleus is slightly oval and contains a single acidophilic nucleolus. CHP shows the nucleus quite distinctly with chromophilic bodies dotted about in the rather reddish purple nucleoplasm. The large celltypes are found mainly on the ventral side of the suboesophageal ganglion. When the extent of vacuolisation is small in these large cells, measurement of the cell bodies is simple and ranges from 30 to 36 p diameter. A basophilic Nissl zone, sometimes very narrow and sometimes diffuse, is often found around the nucleus. The nucleus itself has a diameter of about 8.6 p and contains a single nucleolus. In spite of their size, however, the nuclei of the large cells are not so obvious because they are by no means so chromophilic. Whereas
as those of the small ones the lumina of the vacuoles
NEUROSECRETION
Fig. I. Longitudinal
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157
section of the suboesophageal ganglion of Balnrtus Munoides; granules have been stained with PAF.
brown pigment
.
Fig. 2. Section adjacent to that in Fig. 1; brown pigment granules stained with Schmorl’s k&cyanide reagent.
158
D. B. MCGREGOR
Fig. 3. Supraoesophageal
ganglion
of Cl~thn~uolus srellorlrs staining material.
with a single cell body
Fig. 4. Supraoesophageal
ganglion
of Bola~~s perforatus in which blue-black with CHP.
neurones
contain
packed
with PAF-
globules
stained
~E~ROSECRETiON
Fig. 5. Section through suboesophageal
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159
ganglion of Balums b~~~/fffi~e~stained with PAF.
Fig. 6. Section adjacent to that in Fig. 5; stained with 1~x01 fast blue.
did not usually stain with Sudan black, black granules could often be found singly or in groups around the vacuolar walls. These granules will be discussed later. PASstaining material in the vacuoles may show polarisation artefacts and be removed by amylase, or may be evenly distributed and resistant to the action of amylase. The first substance is most probably glycogen whereas the latter, more sparsely distributed material, is possibly unsaturated lipid. In one specimen of B. ~uluno~~e~a yellowbrown pigment was seen in some vacuoles and fragments of it were also present in the cytoplasm. Attention is drawn to this pigment since it stained with PAP (Fig. 1) and could be confused with neurosecretory granules. This pigment gave a bright blue colour with Schmorl’s ferricyanide reaction (Fig. 2), thus indicating the presence of reducing groups, and also a dark red colour with the Long-Ziehl-Neelsen technique. On the basis of the observed properties it was concluded that the pigment was a lipofuscin (Pearse, 1961). As in the animals previously described (Barnes & Gonor, 1958a, b) all the species examined during the present work possess PAF staining granules. These occur in cell-bodies and occasionally in the axon hillocks of cells scattered throughout the CNS; no pattern in their distribution was evident except in one specimen of ~~t~~~~~l~~
D. B. MCGREGOR
160
stellarus (only
four sectioned)
ganglia
had a partner
staining
granules
where
in a similar
a single cell in one of the supraoesophageal
position
in the other
vary in size, being about 0.2 p diameter
ganglion
in Elminius modestus, 1.O p
in Balanus hameri and 0.2-0.5 ,u in B. balanoides. Generally, stain with the other semi-specific in B. perforatus
where
neurosecretory
phloxinophilic
granules
stains. are
(Fig. 3). The PAF
however,
An exception present,
they do not
to this is found
sometimes
in large
numbers, in the vacuoles of the large cells. When stained with CHP, bluish-black globules of varying sizes may also be found in the ganglia of B. perforatus. It is not certain whether this latter substance is associated with the large cells, but it is clearly associated with the cell-bodies of the small cells (Fig. 4). Ninhydrin-Schiff and mercury-bromophenol blue fail to show any granules which may correspond with the PAF staining ones. Further, the PAF reaction is retained after incubation with either of the endopeptidases. Many red granules are to be seen scattered throughout the CNS when it is stained with PAS or acid-permanganate-Schiff, but these PAS positive granules are removed by prior incubation in amylase for sixteen hours, but not for only two hours. Prolonged exposure to this enzyme does not remove the PAFstaining granules. This suggests that aldehydes are not responsible for the PAF reaction, yet this reaction is reversed after dimedone blocking of any aldehydes produced by oxidation. The alcian blue technique does not stain any structures in nervous tissue in these paraffin sections. On the other hand, in all the species examined CNS granules are stainable with 1~x01 fast blue, even when acid soluble substances are removed, and also with Sudan black B. It is considered that the same granules are involved and that these are probably identical with the PAF positive granules. This conclusion is reached on the grounds that the granules shown by each of these stains are similar in position (compare Figs 5 and 6) and that in specimens where PAF positive granules are abundant so are the others. Examination of B. balanoides between April 1964 and August 1965 showed that individuals with PAF-staining granules in their CNS may be found throughout the year. Individual variations do occur, granules being sparse or perhaps even absent from some whilst they can be found without difficulty in others fixed at the same time. No annual or other long term variation in granule concentration could be clearly detected. Nevertheless, it may be of importance that particularly high concentrations of PAF-staining material were found in April 1964 and again in March 1965; but the results of a single year are an annual cycle. A feature which has not in B. perforatus, of certain direct illumination. These
not enough
on which to base any conclusions
regarding
previously been described in barnacles is the presence, neurones which stand out brightly when observed with cells, packed with refractive material, are found in the
supraoesophageal ganglion and the anterior part of the suboesophageal ganglion, especially at the roots of the nerves. Sometimes the white material can be seen extending some distance along the axons. The location of these cells is not constant, their number and position varying in different individuals (Fig. 7).
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Fig. 7. The typical djstribution of white nerve cells in the freshly dissected CNS of gafa~u~~erforatus: drawn while the CNS was observed under a bright tight; NRVXL., white nerve cell; OES.,oesophagus; SUBOES. G., suboesophageal ganglion; SUPRAOES. G., supraoesophageai ganglion; scale bar = 250,~.
DISCUSSION All neurones secrete - yet not all neurones are called neurosecretory.
The difference is simply one of degree, for ordinary nerve cells secrete synaptic transmitter substances such as acetylcholine, noradrenaline and Shydroxytryptamine. But if some other chemical which is active for a much longer time and over a much greater distance is secreted then the cell may be called a neurosecretory ceil. According to Bern (1963, nerve cells possibly have an endocrine function if granules, droplets or vacuoles are found in tissue sections, regardless of the stain used. If it can be shown that these cytoplasmic inclusions pass through a cycle in which their concentration varies, then it is probable, although by no means certain, that the cells containing them are neurosecretory and have a hormonal function. Any hypothesis of neurosecretion can only be considered proved if the histological evidence can be related to physiological evidence. It is evident that a definite conclusion concerning suspected neurosecretory cells can be reached only if a great deal is known about the physiology of the animal and about its endocrine systems in particular. Because this knowledge is not yet available in sufficient detail for the cirripedes, neurosecretory activity in these animals is, for the present, an unco~rmed possibility.
152
D. B. MCGREGOR
The studies made by Barnes & Gonor (1958a, b) upon several species of barnacles showed that granules stainabIe with PAF are present within all the neurones of all the species examined. The study of the pedunculate Pollicipespolymerus proved to be particularly fruitful. Although they were unable to find evidence of a cycle involving the whole or a particular region of the CNS, Barnes & Gonor (1958a) did describe a cycle of activity through which individual cells seem to pass. In the early part of the cycle, the Nissl zone of large cells stained quite strongly with PAF. Small, PAFstaining granules were sometimes found in the Nissl zone and these appeared to move away from the centre of the cell, enlarging to about 1 p as they did so. At a later stage, the granules had accumulated on the inner side of the peripheral vacuoles, but the Nissl zone and the rest of the cytoplasm did not take up so much stain. Finally, the granuIes usually disappeared, leaving the enlarged peripheral vacuoles, but sometimes amorphous, PAF-staining material remained close to the vacuoles, suggesting that disintegration of the granules had occurred. In the species studied in the present work, however, no correlation could be found between the maturation of the granules and the presence of large vacuoles. Many more cells contain large vacuoles than contain granules and, occasionally, granules may be totally absent from an individual in which the large vacuoles persist. Thus, if indeed there is a correlation, one must assume that, in the operculate species studied here, the process of granule production is rapid in comparison with cellular reconstitution and that this process of granule production becomes extremely rapid at certain times so that the chances of actually finding any cells with PAF-staining granules are slight. Furthermore, the initial stage described for the cycle of granule production in P. polymerus was not observed in the barnacles under consideration here and cells in the intermediate stage, involving outward migration and enlargement of the granules, were seldom encountered. Much more common were cells containing vacuoles and granules or vacuoles alone. Therefore, in these acorn barnacles, there is little evidence-for a neurosecretory cycle like that found in P. polymerus. A possible reason for this difference lies in certain metabolic characteristics of P. polymerus which are rather different from those in Balanus balanoides and EIminius modestus. For example, the age at maturity is several years in PoIlic~pes poIymerus whereas it is only a year in BaIanus baianoides and a few weeks in E~min~us.Furthermore, oxygen uptake, growth rate and almost certainly, the muulting rate are all relatively low in Pollicipes (Barnes, personal commutation). The staining reactions of the cytoplasm& granules in the neurones of the acorn barnacles differ from those obtained with P. polymerus. Whereas the granules of P. polymerus stained with PAS and this ability was not removed by amylase, all PAS-positive granular material was removed from Balanus baianoides neurones by pre-incubation with this enzyme, The PAS reaction on its own does not necessarily indicate that a carbohydrate is present since certain phospholipids may give a red colour (Howe 8c Pearse, 1956). This staining is due to carbon-carbon double bonds in the phospholipids. On the other hand, the granules in Pollicipespolymerus did not stain with Sudan black B, but this may have been due to the fact that the dye was
NEUROSE~RETION
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163
dissolved in ethanol; sudan black B is much more soluble in propylene glycol and stains lipid-containing structures more intensely when this solvent is used. The copper phthalocyanin (1~x01fast blue) technique stains a variety of substances, but provided that calcium is known to be absent there is no difficulty in identifying a positive reaction in the cytoplasm as phospholipid or lipoprotein, though in paraffin sections positive material is more likely to be the latter (Pearse, 1961). Staining with PAP may indicate the presence of any of a number of different substances. For example, Gomori (1950) states that in mammals the following substances and structures are stained: (i) elastic fibres, (ii) mast cells, (iii) chief cells of the gastric mucosa, (iv) B-cells of the pancreatic islets, (v) certain basophils of the anterior pituitary, (vi) some, but not all, kinds of mucin. These staining reactions may be due to (a) a specific reaction with protein, (b) a specific reaction with a mucopolysaccharide or (c) aldehydes produced in the tissue by the oxidizing agent (Bangle, 1954). In P. polymerus, the reactions reported show that aldehydes are definitely produced, but the results obtained from Balanus balanoides are more ambiguous; dimedone certainly blocked the PAF reaction, but aldehydes could not be demonstrated with Schiff’s reagent after oxidation. Thus, it is not possible to say with any certainty that PAF staining is, in this instance, due to the oxidation of ethylenic groups, although this may well be the case in Pollicipes polymerus. CHP-staining granules were found only in Balan~p~r~rat~ out of all the species examined in this and previous work. It is of interest to note that bright white refractive material was also seen in B. p~rforatus; but this material could not be traced for great distances along axons. The presence of this colloid is suggestive either of a high rate of synthetic activity or low rate of mobilisation. Whatever the reason for its concentration, the product is not conveniently localised (in contrast to the situation in the decapods) and hence any role it may have in the metabolism of the barnacle as a whole would be very difficult to ascertain. Neither acid mucopolysaccharides nor proteins have so far been demonstrated although, in view of the strong reactions with lipid stains in these paraffin sections, it is fairly certain that there is a protein moiety to these granules. The significance of the large vacuoles in the neurones is unknown and it is not quite certain that they exist in the living animal. Vacuoles of this type are also a striking feature of the cells in the human supraoptic and paraventricular nuclei (Lundberg, 1958). In the human brain, these vacuoles are usually empty or weakly acidophilic, but some contain blue-black material when stained with CHP. (There is a large taxonomic difference here, but these observations are of interest). Similar vacuoles were found in fixed neurones of portunid crabs (Potter, 1958). In the portunids, however, the vacuoles were not visible in carefully handled living cells. On the other hand, Miyawaki (1956) has demonstrated vacuolations in living cells from the brachyuran Telmessus cheiragonus, but it is not known whether the cells of this species and the portunids examined by Potter are equally sensitive to mechanical disturbances. The staining properties of cellular inclusions from various barnacles are given in Table I where they may be compared with those of neurosecretory cells from other
TABLEI
Locusta migratoria Calliphora erythrocephala Aeschna cyanea Notonecta glauca Perin maxima Gaetice depressus Carcinus maenas Poilicipes polymerus Chthamalus stellatus Elminius modestus Balanus balanoides Balanus perforatus
Man Cat and Dog Rat Nereids
cc..
. . . . .
4-i.. + + +-. +-. -t -+--. .
+
+I-.
+ . . + + + +
+
+ + +
+I-. ++. -it. ++.
-I-+.
+ + -+ f + +
-
-
-.
. .
‘
. .
It -I” -t + t.
. . . . . -
+
5
. . -. --.
-‘r
. * . . .
*
-
-
-
.
+. .
-i-or-+ +.
rfi rtr . . . . f
-
+
.
.
-
-
-
-
_ It
zk t-t-
. -
_ It + .
It k + t
+ + -
. . .
I-
. . .
4 + &: + + + - + i-
+
t-
+
. .
. + + + i .
.
.
.
1
.
. . . f . +
-
3
Lundberg, 1958 Sloper, 1955, 1958 Howe & Pearse, 1956 Defretin, 1955 Arvy & Gabe, 1962 Arvy & Gabe, 1962 Arvy & Gabe, 1962 Arvy & Gabe, 1962 Arvy & Gabe, 1962 Miyawaki, 1960 Rehm, 1959 Barnes & Gonor, 1958a.
Histocbemical reactions obtained from supposed neurosecretory sites in individuals from various animal groups. Tests 1 and 2 are general ‘neurosecretory’ reactions; tests 3 to 8 are for proteins; 9 and 10 are for carbohydrates; 11 to 13 are for lipids; and 14 and 15 are for phospholipids and lipoproteins. CHP; chrome-haematoxylin phloxine; DDD, dihydroxy-dinaphthyldisulphide; PAF, paraldehyde fuchsin; PAS, periodic acid Schiff; PFAAB, performic acid alcian blue; PFAVB, performic acid Victoria blue; $- indicates a strong reaction; & indicates a weak or variable reaction; - indicates no observable reaction.
NEUROSECRETION
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16.5
animals. The single common feature is the ability of the granules to take up PAF after oxidation. B. perforatus is the only barnacle in which the granules are stainable with CHP and even in this species they are not found to contain sulphydryl groups which are demonstrable by the other semi-specific neurosecretory stains, viz. PFAAB, permanganate-alcian blue, PFAVB, reduction DDD, alkaline tetrazolium. Whereas the granules in mammals, insects and malacostracans are mainly composed of protein (i.e. they react with the stains for cystine and cysteine besides alloxan-or ninhydrinSchiff for amino groups, Millon’s reagent for tyrosine, or are removable with trypsin), in barnacles they appear to contain a larger proportion of lipids. Remembering once more the large taxonomic jump involved in such a comparison, Sloper’s (1955) observations on the brain of dogs and cats are particularly relevant to this discussion. In these animals, the PAF and CHP techniques stained material in nervous tissue which definitely was not neurosecretory, i.e. these stains have a wider specificity than was previously supposed. Sloper found that nerve fibres stained lightly and that “coarse cytoplasmic granulesin many neurones, for example in the nuclei of the third cranial nerve, were deeply stained. . . . Similarly granules were demonstrated with Sudan black and the periodic acid-Schiff technique; some also were slightly brown in unstained sections, some reduced ferric ferricyanide to a blue-green pigment without previous reduction of the tissue. These are all properties shared in varying degree by some granules recognised in neurones, some, presumably lipofuscins, and others, socalled ‘glyco-lipid’ granules.” These properties are shared also by at least some of the granules in the neurones of cirripedes. Another line of evidence supporting the suggestion that barnacles do exhibit neurosecretory activity comes from injection experiments (Sandeen 8z Costlow, 1961; Costlow, 1963). By injecting various extracts of barnacle CNS into the fiddler crab, Uca pugilator, they showed that a chromatophorotropin is present in the CNS of several species and that, in Bulanus eburneus, the activity of this substance varies with the stage of the moulting cycle. This evidence is definitely suggestive, but not conclusive, of neurosecretory activity because it is not known whether or not the substance leaves the nervous system. In the prawn, Palae~one~es, for example, chromatophorotropins are present in the CNS, but there is little indication that they leave the CNS and normally participate in colour change controf (Kleinholz, 1961). The steric specificity required of tropic substances by chromatophores is not known. It could be that some constituent (not a hormone) of the CNS is responsible for the observed colour changes, its concentration varying with the overall activity of the organ. Furthermore, in Bulunus eburneus the substance cannot function as a chromatophorotropin because cirripedes do not possess chromatophores. Nevertheless, the injection experiments made by Sandeen & Costlow and the histological studies by Barnes & Gonor do point to the possibility that there are neurosecretory processess in barnacles; but the situation is still far from clear and further studies are obviously needed, particular attention being paid to the control of the moulting cycle.
166
D. B. MCGREGOR ACKNOWLEDGEMENTS
I am grateful to the National Environment Research Council who financed this work. I also wish to express my thanks to Professor D. R. Arthur for allowing this work to be carried out in the Department of Zoology, King’s College, London, Dr. S. K. Eltringham for supervising the work and discussing the results and Dr. H. Barnes for reading the manuscript and offering suggestions for its improvement. REFERENCES ARVY, L. & M. GABE, 1962. Histochemistry of insect neurosecretion. In, Neurosecretion, edited by H. Heller & R. B. Clark, Academic Press, London, pp. 331-344. BANGLE,R., 1954. Gomori’s paraldehyde-fuchsin stain. I. Physico-chemical and staining properties of the dye. J. Histochem. Cytochem., Vol. 2, pp. 291-299. BARGMANN,W., 1949. Ueber die neurosekretorische Verkniipfung von Hypothalamus und Neurohypophyse. Z. Zellforsch. mikrosk. Anat., Bd 34, S. 610-634. BARNES,H. & J. J. GONOR, 1958a. Neurosecretory cells in the cirripede PolZicipes polymerus J. B. Sowerby. J. mar. Res., Vol. 17, pp. 81-102. BARNES,H. & J. J. GONOR, 1958b. Neurosecretory cells in some cirripedes. Nature, Lond., Vol. 181, p. 194 only. BERN, H. A., 1962. The properties of neurosecretory cells. Gen. Comp. Endocrinol., Suppl. 1, pp. 117-132. BONHAG,P. F., 1955. Histochemical studies of the ovarian nurse tissues and oocytes of the milkweed bug, Oncopeltus fasciatus Dallas. I. Cytology, nucleic acids and carbohydrates. J. Morph., Vol. 96, pp. 51-56. CHIFFELLE,T. L. & F. A. Purr, 1951. Propylene and ethylene glycol as solvents for Sudan IV and Sudan black B. Stain Technol., Vol. 26, pp. 51-56. DEFRETIN,R., 1955. Recherches cytologiques et histochimiques sur le systeme nerveux des nCrCidiens. La neurosCcr&ion des polyosides et ses rapports avec l’kpitoquie. Archs. Zool. exp. g&z., T. 92, pp. 73-140. COSTLOW,J. D., Jr, 1963. Moulting and cyclic activity in chromatophorotropins of the CNS of the barnacle Balanus eburneus. Biol. Bull. mar. biol. Lab., Woods Hole, Vol. 124, pp. 254-261. GABE, M., 1953. Sur quelques applications de la coloration par la fuchsine-paraldehyde. Bull. Microsc. appl., T. 3, pp. 153-162. GOMORI,G., 1950. Aldehyde-fuchsin: a new stain for elastic tissue. Amer. J. Clin. Pathol., Vol. 20, pp. 665-666. HOWE, A. & A. G. E. PEARSE,1956. A histochemical investigation of neurosecretory substance in the rat. J. Histochem. Cytochem., Vol. 4, pp. 561-569. KLEINHOLZ,L. H., 1961. Pigment effecters. In, The physiology of crustacea, Vol. ZZ, edited by T. H. Waterman, Academic Press, New York, pp. 141-142. KLUVER, H. & E. BARRERA,1953. A method for the combined staining of cells and fibres in the nervous system. J. Neuropath. exp. Neural., Vol. 12, pp. 400-403. LUNDBERG,P. O., 1958. Neurosecretory and related phenomena in the hypothalamus and pituitary of man. In, Zweites Znternationales Symposium iiber Neurosekretion, edited by W. Bargmann, B. HanstrBm, B. & E. Scharrer, Springer-Verlag, Berlin, pp. 13-17. MIYAWAKI,M., 1956. Cytological and cytochemical studies on the neurosecretory cells of a Brachyuran Telmessus cheiragonus (Tilesius). J. Fat. Sci. Hokkaido Univ., Ser. VI, Zool., Vol. 12, pp. 516-520. MIYAWAKI,M., 1960. Studies on the cytoplasmic globules in nerve cells of the crabs, Gaetice depressus and Potamon dehaani. Kumamoto J. Sci., Vol. 5, pp. l--10. PEARSE,A. G. E., 1961. Histochemistry, Churchill, London, 998 pp. POTTER, D. D., 1958. Observations on the neurosecretory system of portunid crabs. In, Zweites Znternationales Symposium iiber Neurosekretion, edited by W. Bargmann, B. Hanstram, B. & E. Scharrer, Springer-Verlag, Berlin, pp. 113-l 18.
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M., 1959. Observations on the locahsation and chemical canst~tution of neurosecretory material in the nerve terminals of Car&us maenas. Acta histochem., Vol. 7, pp. 88-106. SANDEEN, M. I. & J. D. COSTLOW,Jr., 1961. The presence of decapod pigment activating substances in the CNS of representative cirripedes. Biol. Bull. mar. biol. Lab., Woods Hole, Vot. 120, pp. 192-205. SLOPER, J. C., 1955. Hypothalamic neurosecretion in the dog and cat, with particular reference to the identification of neurosecretory material with posterior lobe hormone. J. Anat., Vol. 89, pp. 301316. SLOPER, J. C., 1958. The application of newer histochemical and isotope techniques for the location of protein bound cystine and cysteine to the study of hypothalamic neurosecretion in normal and pathological conditions. In, Zweites internationales Symposium iiber Neurosekretion, edited by W. Bargmann, B. Hanstrom, B. & E. Scharrer, Springer-Verlag, Berlin, pp. 20-25. STEEDMAN, H. F., 1950. Alcian blue 8GS: a new stain for mucin. Q. Ji microsc. Sci., Vol. 91, pp. 471-419. YASUMA, A. & T. ICHIKAWA, 1953. Ni~hydri~-Sch~ and al~oxan-Schiff staining. J. Lab. clin. Med., Vol. 41, pp. 296-299.
1, pp. 154-l 69; North-Holland
THE NEUROSECRETORY
Publishing Company,
Amsterdam
CELLS OF BARNACLES
D. B. MCGREGOR 1 Departmentof Zoofogy,King’s College, London, Ettglmd Abstract: The central nervous systems of several species of acorn barnacles have been examined for the presence of so-called neurosecretory granules. PAF-staining granules were found in all the species but only in Bulunus perfbratus could CHP-staining granules be found. In this species also, colloidal accumulations were found which were reminiscent of the refractive material which is so characteristic of the sinus glands and other neurosecretory centres in malacostracans. The granules also stained with Sudan black, when dissolved in propylene glycol, and 1~x01 fast blue, but the use of stains for proteins was unsuccessful. This is rather different from the results obtained with ne~rosecretory granules from a variety of other sources. The staining characteristics of the granules from these acorn barnacles were also slightly different from those reported for the stalked barnacle, Pollicipespolymerus. A long-term cycle of PAF-staining granule concentrations, although looked for in Balanus baianoides, could not be found. It is concluded that there is evidence suggestive of neurosecretory activity but that neither the histological nor the physiological approaches have, as yet, provided unequivocal answers.
INTRODUCTION
Apart from the decapods, the crustaceans have received scant attention from physiologists engaged on neurosecretory studies. Within the last ten years, however, several attempts have been made to apply the methods so suc~ssfully used with decapods to the entomostracans. Some success has been achieved, particularly with the cirripedes. In this paper the results are reported of a histological study on the nerve cells of various barnacles, and their bearing upon the problem of neurosecretion in these animals is discussed. MATERIALS AND METHODS
Most of this work has been done on Balanus balanoides and B. perforatus; in addition B. crenatus, B. humeri, B. spongicola, Chthamalus stellatus, Verruca stroemia, and Elminius modestus have been investigated. If the central nervous system (CNS) were to be examined fresh, the soft part of the animal was removed from its shell and the dissection carried out with the live animal immersed in sea water. The animal was held in its normal position by one pin through the anterior part of the prosoma and another bent over the cirri. By carefully removing the mouth cone, the CNS, which lies directly beneath it, could be seen in a bright light. The long nerves from the s~boesophageal ganglion leading to the cirri and to 1 Present address: Flour-Millers’
and Bakers’
Research
England 154
Association,
Old London Road, St. Album,
NEUROSE~RETIO~
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155
the prosoma as well as those from the tiny supraoesophogeal ganglion were cut and the principal ganglia eased away from the surrounding tissue. When the material was to be examined histologically, the soft parts were removed from the shell and fixed in alcoholic Bouin’s fluid or in neutral buffered formalin. Following dehydration in cellosolve, the specimens were cleared in benzene and then infiltrated and embedded in paraffin wax (m.p. 52 “C). Sections were cut at 7~, smoothed out by floating on warm water and allowed to dry on clean glass slides. No affixative was used. The staining techniques used were as follows:
(i) Periodic acid-Schiff (PAS) for glycolic groups (Pearse, 1961, after ~otchkiss). (ii) Amylase-PAS for distinguishing glycogen from other glycol-containing substances. (iii) Amylase-paraldehyde fuchsin to remove glycogen before staining neurosecretory substances. (iv) Alcian blue for acid mucopolysaccharides (Steedman, 1950). Lipids
(i) Sudan black B in propylene glycol for neutral fats and lipoproteins (Chiffelle & Putt, 1951). (ii) Luxoi fast blue (a copper phthalocyanin~ for phospholipids and lipoproteins (Kluver & Barrera, 1953). Proteins
(i) Ninhydrin-Schiff for protein bound -NH, groups (Yasuma & Ichikawa, 1953). (ii) Performic acid - Victoria blue (PFAVB) for sulphydryl groups (Humberstone, personal communication). This is a semi-specific stain for neurosecretory substances. (iii) Mercury-bromophenol blue for proteins (Bonhag, 1955). (iv) Pepsin-paraldehyde fuchsin. Incubation as recommended by Pearse (1961). (v) Trypsin-paraldehyde fuchsin. Incubation as recommended by Pearse (1961).
(i) Chrome haematoxylin-phlo~ne B (CHP) ~Bargmann, 1949). This is a semispecific stain for neurosecretory substances. (ii) Paraldehyde fuchsin (PAF) (Gabe, 1953). Used with preliminary oxidation as in the CHP technique, this is a semi-specific stain for neurosecretory material. (iii) Acid-permanganate-Schiff for the demonstration of aldehydes exposed by the oxidation procedures used for CHP and PAF staining. (iv) Acid-permanganate-diedone-PAF to block aldehydes produced in the oxidation procedure. Blocking with dimedone as recommended by Pearse (1961). (v) Acid-permanganate-dimedone-Schiff. Used to check that the blockage of aldehydes by dimedone had been successful.
156
D. B. MCGREGOR
(vi) Schmorl’s ferricyanide for lipofuscin, melanin, and argentaffin granules (Pearse, 1961). (vii) Long-Ziehl-Ne~ls~n method for acid-fast lipofuscins and lipoproteins (Pearse, 1961). In order to investigate the possibility of a long term cycle of secretory activity, Balunus balunoides was sampled at one to two weekly intervals from both Brighton and Bradwell-on-Sea between April 1964 and August 1965. From each sample random sections through the CNS of about six individuals were stained and examined. RESULTS
The general histology of the CNS has been described (Barnes & Gonor, 1958a). The outstanding feature of the neurones is the numerous vacuoles of varying sizes which are always present peripherally in the cell-bodies and which may fill them to such an extent that the cytoplasm is reduced to a few thin strands surrounding the vacuoles and the cell nucleus. These vacuoles may be partly fixation artefacts but large vacuoles are also visible in nerve cells teased from fresh nervous tissues of B. balanoides and their contents can be seen oozing out of the cell when squashed. Nevertheless, the shock caused by the dissection procedure may be enough to produce great morphological changes in the cytoplasm. As with the species described by Barnes & Gonor, the present species had two kinds of cell in their ganglia. The cells of B. perfuratus will be described in detail but those of the other species examined are similarly differentiated into two types. Cell dimensions are di~cult to determine accurately because the vacuoles in the cytoplasm distort the cell outline and often make it impossible to distinguish between the cytoplasm of one cell and that of an adjacent one. The nuclei of the small cell type average 4.2 p in diameter and are found in all parts of the ganglia where cell bodies occur. In the fused suboesophageal ganglion these small nuclei are found mainly on the dorsal side although they are also present in the ventral, lateral and central parts of the ganglion. The cell bodies of these small neurones average 9.5 I( diameter; hence there is very little cytoplasm in the cell bodies and the structured cytoplasm (in sectioned material) may be further reduced by the presence of vacuoles. No basophilic Nissl zone was ever observed. The nucleus is slightly oval and contains a single acidophilic nucleolus. CHP shows the nucleus quite distinctly with chromophilic bodies dotted about in the rather reddish purple nucleoplasm. The large celltypes are found mainly on the ventral side of the suboesophageal ganglion. When the extent of vacuolisation is small in these large cells, measurement of the cell bodies is simple and ranges from 30 to 36 p diameter. A basophilic Nissl zone, sometimes very narrow and sometimes diffuse, is often found around the nucleus. The nucleus itself has a diameter of about 8.6 p and contains a single nucleolus. In spite of their size, however, the nuclei of the large cells are not so obvious because they are by no means so chromophilic. Whereas
as those of the small ones the lumina of the vacuoles
NEUROSECRETION
Fig. I. Longitudinal
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157
section of the suboesophageal ganglion of Balnrtus Munoides; granules have been stained with PAF.
brown pigment
.
Fig. 2. Section adjacent to that in Fig. 1; brown pigment granules stained with Schmorl’s k&cyanide reagent.
158
D. B. MCGREGOR
Fig. 3. Supraoesophageal
ganglion
of Cl~thn~uolus srellorlrs staining material.
with a single cell body
Fig. 4. Supraoesophageal
ganglion
of Bola~~s perforatus in which blue-black with CHP.
neurones
contain
packed
with PAF-
globules
stained
~E~ROSECRETiON
Fig. 5. Section through suboesophageal
IN BARNACLES
159
ganglion of Balums b~~~/fffi~e~stained with PAF.
Fig. 6. Section adjacent to that in Fig. 5; stained with 1~x01 fast blue.
did not usually stain with Sudan black, black granules could often be found singly or in groups around the vacuolar walls. These granules will be discussed later. PASstaining material in the vacuoles may show polarisation artefacts and be removed by amylase, or may be evenly distributed and resistant to the action of amylase. The first substance is most probably glycogen whereas the latter, more sparsely distributed material, is possibly unsaturated lipid. In one specimen of B. ~uluno~~e~a yellowbrown pigment was seen in some vacuoles and fragments of it were also present in the cytoplasm. Attention is drawn to this pigment since it stained with PAP (Fig. 1) and could be confused with neurosecretory granules. This pigment gave a bright blue colour with Schmorl’s ferricyanide reaction (Fig. 2), thus indicating the presence of reducing groups, and also a dark red colour with the Long-Ziehl-Neelsen technique. On the basis of the observed properties it was concluded that the pigment was a lipofuscin (Pearse, 1961). As in the animals previously described (Barnes & Gonor, 1958a, b) all the species examined during the present work possess PAF staining granules. These occur in cell-bodies and occasionally in the axon hillocks of cells scattered throughout the CNS; no pattern in their distribution was evident except in one specimen of ~~t~~~~~l~~
D. B. MCGREGOR
160
stellarus (only
four sectioned)
ganglia
had a partner
staining
granules
where
in a similar
a single cell in one of the supraoesophageal
position
in the other
vary in size, being about 0.2 p diameter
ganglion
in Elminius modestus, 1.O p
in Balanus hameri and 0.2-0.5 ,u in B. balanoides. Generally, stain with the other semi-specific in B. perforatus
where
neurosecretory
phloxinophilic
granules
stains. are
(Fig. 3). The PAF
however,
An exception present,
they do not
to this is found
sometimes
in large
numbers, in the vacuoles of the large cells. When stained with CHP, bluish-black globules of varying sizes may also be found in the ganglia of B. perforatus. It is not certain whether this latter substance is associated with the large cells, but it is clearly associated with the cell-bodies of the small cells (Fig. 4). Ninhydrin-Schiff and mercury-bromophenol blue fail to show any granules which may correspond with the PAF staining ones. Further, the PAF reaction is retained after incubation with either of the endopeptidases. Many red granules are to be seen scattered throughout the CNS when it is stained with PAS or acid-permanganate-Schiff, but these PAS positive granules are removed by prior incubation in amylase for sixteen hours, but not for only two hours. Prolonged exposure to this enzyme does not remove the PAFstaining granules. This suggests that aldehydes are not responsible for the PAF reaction, yet this reaction is reversed after dimedone blocking of any aldehydes produced by oxidation. The alcian blue technique does not stain any structures in nervous tissue in these paraffin sections. On the other hand, in all the species examined CNS granules are stainable with 1~x01 fast blue, even when acid soluble substances are removed, and also with Sudan black B. It is considered that the same granules are involved and that these are probably identical with the PAF positive granules. This conclusion is reached on the grounds that the granules shown by each of these stains are similar in position (compare Figs 5 and 6) and that in specimens where PAF positive granules are abundant so are the others. Examination of B. balanoides between April 1964 and August 1965 showed that individuals with PAF-staining granules in their CNS may be found throughout the year. Individual variations do occur, granules being sparse or perhaps even absent from some whilst they can be found without difficulty in others fixed at the same time. No annual or other long term variation in granule concentration could be clearly detected. Nevertheless, it may be of importance that particularly high concentrations of PAF-staining material were found in April 1964 and again in March 1965; but the results of a single year are an annual cycle. A feature which has not in B. perforatus, of certain direct illumination. These
not enough
on which to base any conclusions
regarding
previously been described in barnacles is the presence, neurones which stand out brightly when observed with cells, packed with refractive material, are found in the
supraoesophageal ganglion and the anterior part of the suboesophageal ganglion, especially at the roots of the nerves. Sometimes the white material can be seen extending some distance along the axons. The location of these cells is not constant, their number and position varying in different individuals (Fig. 7).
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161
Fig. 7. The typical djstribution of white nerve cells in the freshly dissected CNS of gafa~u~~erforatus: drawn while the CNS was observed under a bright tight; NRVXL., white nerve cell; OES.,oesophagus; SUBOES. G., suboesophageal ganglion; SUPRAOES. G., supraoesophageai ganglion; scale bar = 250,~.
DISCUSSION All neurones secrete - yet not all neurones are called neurosecretory.
The difference is simply one of degree, for ordinary nerve cells secrete synaptic transmitter substances such as acetylcholine, noradrenaline and Shydroxytryptamine. But if some other chemical which is active for a much longer time and over a much greater distance is secreted then the cell may be called a neurosecretory ceil. According to Bern (1963, nerve cells possibly have an endocrine function if granules, droplets or vacuoles are found in tissue sections, regardless of the stain used. If it can be shown that these cytoplasmic inclusions pass through a cycle in which their concentration varies, then it is probable, although by no means certain, that the cells containing them are neurosecretory and have a hormonal function. Any hypothesis of neurosecretion can only be considered proved if the histological evidence can be related to physiological evidence. It is evident that a definite conclusion concerning suspected neurosecretory cells can be reached only if a great deal is known about the physiology of the animal and about its endocrine systems in particular. Because this knowledge is not yet available in sufficient detail for the cirripedes, neurosecretory activity in these animals is, for the present, an unco~rmed possibility.
152
D. B. MCGREGOR
The studies made by Barnes & Gonor (1958a, b) upon several species of barnacles showed that granules stainabIe with PAF are present within all the neurones of all the species examined. The study of the pedunculate Pollicipespolymerus proved to be particularly fruitful. Although they were unable to find evidence of a cycle involving the whole or a particular region of the CNS, Barnes & Gonor (1958a) did describe a cycle of activity through which individual cells seem to pass. In the early part of the cycle, the Nissl zone of large cells stained quite strongly with PAF. Small, PAFstaining granules were sometimes found in the Nissl zone and these appeared to move away from the centre of the cell, enlarging to about 1 p as they did so. At a later stage, the granules had accumulated on the inner side of the peripheral vacuoles, but the Nissl zone and the rest of the cytoplasm did not take up so much stain. Finally, the granuIes usually disappeared, leaving the enlarged peripheral vacuoles, but sometimes amorphous, PAF-staining material remained close to the vacuoles, suggesting that disintegration of the granules had occurred. In the species studied in the present work, however, no correlation could be found between the maturation of the granules and the presence of large vacuoles. Many more cells contain large vacuoles than contain granules and, occasionally, granules may be totally absent from an individual in which the large vacuoles persist. Thus, if indeed there is a correlation, one must assume that, in the operculate species studied here, the process of granule production is rapid in comparison with cellular reconstitution and that this process of granule production becomes extremely rapid at certain times so that the chances of actually finding any cells with PAF-staining granules are slight. Furthermore, the initial stage described for the cycle of granule production in P. polymerus was not observed in the barnacles under consideration here and cells in the intermediate stage, involving outward migration and enlargement of the granules, were seldom encountered. Much more common were cells containing vacuoles and granules or vacuoles alone. Therefore, in these acorn barnacles, there is little evidence-for a neurosecretory cycle like that found in P. polymerus. A possible reason for this difference lies in certain metabolic characteristics of P. polymerus which are rather different from those in Balanus balanoides and EIminius modestus. For example, the age at maturity is several years in PoIlic~pes poIymerus whereas it is only a year in BaIanus baianoides and a few weeks in E~min~us.Furthermore, oxygen uptake, growth rate and almost certainly, the muulting rate are all relatively low in Pollicipes (Barnes, personal commutation). The staining reactions of the cytoplasm& granules in the neurones of the acorn barnacles differ from those obtained with P. polymerus. Whereas the granules of P. polymerus stained with PAS and this ability was not removed by amylase, all PAS-positive granular material was removed from Balanus baianoides neurones by pre-incubation with this enzyme, The PAS reaction on its own does not necessarily indicate that a carbohydrate is present since certain phospholipids may give a red colour (Howe 8c Pearse, 1956). This staining is due to carbon-carbon double bonds in the phospholipids. On the other hand, the granules in Pollicipespolymerus did not stain with Sudan black B, but this may have been due to the fact that the dye was
NEUROSE~RETION
IN BARNACLES
163
dissolved in ethanol; sudan black B is much more soluble in propylene glycol and stains lipid-containing structures more intensely when this solvent is used. The copper phthalocyanin (1~x01fast blue) technique stains a variety of substances, but provided that calcium is known to be absent there is no difficulty in identifying a positive reaction in the cytoplasm as phospholipid or lipoprotein, though in paraffin sections positive material is more likely to be the latter (Pearse, 1961). Staining with PAP may indicate the presence of any of a number of different substances. For example, Gomori (1950) states that in mammals the following substances and structures are stained: (i) elastic fibres, (ii) mast cells, (iii) chief cells of the gastric mucosa, (iv) B-cells of the pancreatic islets, (v) certain basophils of the anterior pituitary, (vi) some, but not all, kinds of mucin. These staining reactions may be due to (a) a specific reaction with protein, (b) a specific reaction with a mucopolysaccharide or (c) aldehydes produced in the tissue by the oxidizing agent (Bangle, 1954). In P. polymerus, the reactions reported show that aldehydes are definitely produced, but the results obtained from Balanus balanoides are more ambiguous; dimedone certainly blocked the PAF reaction, but aldehydes could not be demonstrated with Schiff’s reagent after oxidation. Thus, it is not possible to say with any certainty that PAF staining is, in this instance, due to the oxidation of ethylenic groups, although this may well be the case in Pollicipes polymerus. CHP-staining granules were found only in Balan~p~r~rat~ out of all the species examined in this and previous work. It is of interest to note that bright white refractive material was also seen in B. p~rforatus; but this material could not be traced for great distances along axons. The presence of this colloid is suggestive either of a high rate of synthetic activity or low rate of mobilisation. Whatever the reason for its concentration, the product is not conveniently localised (in contrast to the situation in the decapods) and hence any role it may have in the metabolism of the barnacle as a whole would be very difficult to ascertain. Neither acid mucopolysaccharides nor proteins have so far been demonstrated although, in view of the strong reactions with lipid stains in these paraffin sections, it is fairly certain that there is a protein moiety to these granules. The significance of the large vacuoles in the neurones is unknown and it is not quite certain that they exist in the living animal. Vacuoles of this type are also a striking feature of the cells in the human supraoptic and paraventricular nuclei (Lundberg, 1958). In the human brain, these vacuoles are usually empty or weakly acidophilic, but some contain blue-black material when stained with CHP. (There is a large taxonomic difference here, but these observations are of interest). Similar vacuoles were found in fixed neurones of portunid crabs (Potter, 1958). In the portunids, however, the vacuoles were not visible in carefully handled living cells. On the other hand, Miyawaki (1956) has demonstrated vacuolations in living cells from the brachyuran Telmessus cheiragonus, but it is not known whether the cells of this species and the portunids examined by Potter are equally sensitive to mechanical disturbances. The staining properties of cellular inclusions from various barnacles are given in Table I where they may be compared with those of neurosecretory cells from other
TABLEI
Locusta migratoria Calliphora erythrocephala Aeschna cyanea Notonecta glauca Perin maxima Gaetice depressus Carcinus maenas Poilicipes polymerus Chthamalus stellatus Elminius modestus Balanus balanoides Balanus perforatus
Man Cat and Dog Rat Nereids
cc..
. . . . .
4-i.. + + +-. +-. -t -+--. .
+
+I-.
+ . . + + + +
+
+ + +
+I-. ++. -it. ++.
-I-+.
+ + -+ f + +
-
-
-.
. .
‘
. .
It -I” -t + t.
. . . . . -
+
5
. . -. --.
-‘r
. * . . .
*
-
-
-
.
+. .
-i-or-+ +.
rfi rtr . . . . f
-
+
.
.
-
-
-
-
_ It
zk t-t-
. -
_ It + .
It k + t
+ + -
. . .
I-
. . .
4 + &: + + + - + i-
+
t-
+
. .
. + + + i .
.
.
.
1
.
. . . f . +
-
3
Lundberg, 1958 Sloper, 1955, 1958 Howe & Pearse, 1956 Defretin, 1955 Arvy & Gabe, 1962 Arvy & Gabe, 1962 Arvy & Gabe, 1962 Arvy & Gabe, 1962 Arvy & Gabe, 1962 Miyawaki, 1960 Rehm, 1959 Barnes & Gonor, 1958a.
Histocbemical reactions obtained from supposed neurosecretory sites in individuals from various animal groups. Tests 1 and 2 are general ‘neurosecretory’ reactions; tests 3 to 8 are for proteins; 9 and 10 are for carbohydrates; 11 to 13 are for lipids; and 14 and 15 are for phospholipids and lipoproteins. CHP; chrome-haematoxylin phloxine; DDD, dihydroxy-dinaphthyldisulphide; PAF, paraldehyde fuchsin; PAS, periodic acid Schiff; PFAAB, performic acid alcian blue; PFAVB, performic acid Victoria blue; $- indicates a strong reaction; & indicates a weak or variable reaction; - indicates no observable reaction.
NEUROSECRETION
IN BARNACLES
16.5
animals. The single common feature is the ability of the granules to take up PAF after oxidation. B. perforatus is the only barnacle in which the granules are stainable with CHP and even in this species they are not found to contain sulphydryl groups which are demonstrable by the other semi-specific neurosecretory stains, viz. PFAAB, permanganate-alcian blue, PFAVB, reduction DDD, alkaline tetrazolium. Whereas the granules in mammals, insects and malacostracans are mainly composed of protein (i.e. they react with the stains for cystine and cysteine besides alloxan-or ninhydrinSchiff for amino groups, Millon’s reagent for tyrosine, or are removable with trypsin), in barnacles they appear to contain a larger proportion of lipids. Remembering once more the large taxonomic jump involved in such a comparison, Sloper’s (1955) observations on the brain of dogs and cats are particularly relevant to this discussion. In these animals, the PAF and CHP techniques stained material in nervous tissue which definitely was not neurosecretory, i.e. these stains have a wider specificity than was previously supposed. Sloper found that nerve fibres stained lightly and that “coarse cytoplasmic granulesin many neurones, for example in the nuclei of the third cranial nerve, were deeply stained. . . . Similarly granules were demonstrated with Sudan black and the periodic acid-Schiff technique; some also were slightly brown in unstained sections, some reduced ferric ferricyanide to a blue-green pigment without previous reduction of the tissue. These are all properties shared in varying degree by some granules recognised in neurones, some, presumably lipofuscins, and others, socalled ‘glyco-lipid’ granules.” These properties are shared also by at least some of the granules in the neurones of cirripedes. Another line of evidence supporting the suggestion that barnacles do exhibit neurosecretory activity comes from injection experiments (Sandeen 8z Costlow, 1961; Costlow, 1963). By injecting various extracts of barnacle CNS into the fiddler crab, Uca pugilator, they showed that a chromatophorotropin is present in the CNS of several species and that, in Bulanus eburneus, the activity of this substance varies with the stage of the moulting cycle. This evidence is definitely suggestive, but not conclusive, of neurosecretory activity because it is not known whether or not the substance leaves the nervous system. In the prawn, Palae~one~es, for example, chromatophorotropins are present in the CNS, but there is little indication that they leave the CNS and normally participate in colour change controf (Kleinholz, 1961). The steric specificity required of tropic substances by chromatophores is not known. It could be that some constituent (not a hormone) of the CNS is responsible for the observed colour changes, its concentration varying with the overall activity of the organ. Furthermore, in Bulunus eburneus the substance cannot function as a chromatophorotropin because cirripedes do not possess chromatophores. Nevertheless, the injection experiments made by Sandeen & Costlow and the histological studies by Barnes & Gonor do point to the possibility that there are neurosecretory processess in barnacles; but the situation is still far from clear and further studies are obviously needed, particular attention being paid to the control of the moulting cycle.
166
D. B. MCGREGOR ACKNOWLEDGEMENTS
I am grateful to the National Environment Research Council who financed this work. I also wish to express my thanks to Professor D. R. Arthur for allowing this work to be carried out in the Department of Zoology, King’s College, London, Dr. S. K. Eltringham for supervising the work and discussing the results and Dr. H. Barnes for reading the manuscript and offering suggestions for its improvement. REFERENCES ARVY, L. & M. GABE, 1962. Histochemistry of insect neurosecretion. In, Neurosecretion, edited by H. Heller & R. B. Clark, Academic Press, London, pp. 331-344. BANGLE,R., 1954. Gomori’s paraldehyde-fuchsin stain. I. Physico-chemical and staining properties of the dye. J. Histochem. Cytochem., Vol. 2, pp. 291-299. BARGMANN,W., 1949. Ueber die neurosekretorische Verkniipfung von Hypothalamus und Neurohypophyse. Z. Zellforsch. mikrosk. Anat., Bd 34, S. 610-634. BARNES,H. & J. J. GONOR, 1958a. Neurosecretory cells in the cirripede PolZicipes polymerus J. B. Sowerby. J. mar. Res., Vol. 17, pp. 81-102. BARNES,H. & J. J. GONOR, 1958b. Neurosecretory cells in some cirripedes. Nature, Lond., Vol. 181, p. 194 only. BERN, H. A., 1962. The properties of neurosecretory cells. Gen. Comp. Endocrinol., Suppl. 1, pp. 117-132. BONHAG,P. F., 1955. Histochemical studies of the ovarian nurse tissues and oocytes of the milkweed bug, Oncopeltus fasciatus Dallas. I. Cytology, nucleic acids and carbohydrates. J. Morph., Vol. 96, pp. 51-56. CHIFFELLE,T. L. & F. A. Purr, 1951. Propylene and ethylene glycol as solvents for Sudan IV and Sudan black B. Stain Technol., Vol. 26, pp. 51-56. DEFRETIN,R., 1955. Recherches cytologiques et histochimiques sur le systeme nerveux des nCrCidiens. La neurosCcr&ion des polyosides et ses rapports avec l’kpitoquie. Archs. Zool. exp. g&z., T. 92, pp. 73-140. COSTLOW,J. D., Jr, 1963. Moulting and cyclic activity in chromatophorotropins of the CNS of the barnacle Balanus eburneus. Biol. Bull. mar. biol. Lab., Woods Hole, Vol. 124, pp. 254-261. GABE, M., 1953. Sur quelques applications de la coloration par la fuchsine-paraldehyde. Bull. Microsc. appl., T. 3, pp. 153-162. GOMORI,G., 1950. Aldehyde-fuchsin: a new stain for elastic tissue. Amer. J. Clin. Pathol., Vol. 20, pp. 665-666. HOWE, A. & A. G. E. PEARSE,1956. A histochemical investigation of neurosecretory substance in the rat. J. Histochem. Cytochem., Vol. 4, pp. 561-569. KLEINHOLZ,L. H., 1961. Pigment effecters. In, The physiology of crustacea, Vol. ZZ, edited by T. H. Waterman, Academic Press, New York, pp. 141-142. KLUVER, H. & E. BARRERA,1953. A method for the combined staining of cells and fibres in the nervous system. J. Neuropath. exp. Neural., Vol. 12, pp. 400-403. LUNDBERG,P. O., 1958. Neurosecretory and related phenomena in the hypothalamus and pituitary of man. In, Zweites Znternationales Symposium iiber Neurosekretion, edited by W. Bargmann, B. HanstrBm, B. & E. Scharrer, Springer-Verlag, Berlin, pp. 13-17. MIYAWAKI,M., 1956. Cytological and cytochemical studies on the neurosecretory cells of a Brachyuran Telmessus cheiragonus (Tilesius). J. Fat. Sci. Hokkaido Univ., Ser. VI, Zool., Vol. 12, pp. 516-520. MIYAWAKI,M., 1960. Studies on the cytoplasmic globules in nerve cells of the crabs, Gaetice depressus and Potamon dehaani. Kumamoto J. Sci., Vol. 5, pp. l--10. PEARSE,A. G. E., 1961. Histochemistry, Churchill, London, 998 pp. POTTER, D. D., 1958. Observations on the neurosecretory system of portunid crabs. In, Zweites Znternationales Symposium iiber Neurosekretion, edited by W. Bargmann, B. Hanstram, B. & E. Scharrer, Springer-Verlag, Berlin, pp. 113-l 18.
NEUROSECR~TION REHM,
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