GENERAL AND COMPAR4TIVE ENDOCRINOLOGY 22, 454458 (1974)

A Histochemical investigation of Neurosecretion

in Enchytraeus albidus (Enchytraeidae)

STEFANO BIANCHI

Institute of Zoology of the University of Naples, Naples, Italy

Received July 30, 1973

The histochemistry of the neurosecretions in the cerebral and subesophageal ganglia of Enchytraeus is examined. The secretions of Q and U cells contain cystine. The P cell secretions are characterized by a high content of lipids detect- able in paraffin sections.

In Enchytraeus albidus the cerebral gan- glion contains two types of neurosecretory neuron: P cells and & cells (Deuse-Zim- mermann, 1960). P cells are eight in num- ber and lie in t.he caudal part of the cerebral ganglion. Their secretory product stains weakly with paraldehyde fuchsin (PF) but exhibits no affinity for chrome hematoxylin in the Gomori method. It can be detected in the axons. Q cells are two in number and lie in the laterocaudal part of the cerebral ganglion. Their secretory product takes up chrome hematoxylin and PF. It can be detected in the proximal parts of the axons (Deuse-Zimmermann, 1960). In the subesophageal ganglion of Enchytraeus are present two neurosecre- tory cells, the U cells (Gersch and Ude, 1967). U cells whose morphological char- acteristics are similar to those of Q cells, contain secretion granules staining with PF and chrome hematoxylin. The granules are present also in the axons (Deuse-Zim- mermann, 1960).

We know little of the histochemical characteristics of neurosecretory cells in Enchytraeus. The staining with Sudan III and with osmic acid vapor indicates that the secretory product of P cells is rich in formed lipid inclusions (Deuse-Zimmer- mann, 1960).

It therefore seemed opportune to under- take a thorough investigation on the chem- ical nature of neurosecretory material of

the neurosecretory cells in Enchytraeus albidus.

MATERIAL AND METHODS

Specimens of adult Enchytraeus were taken from the aquarium of Bologna at regular monthly intervals. The observation reported here were made from January through December. The an- terior body segments including the cerebral ganglion and the subesophageal ganglion were fixed in the appropriate fixative. For the Sudan black technique, the material was fixed W-24 hr in Helly’s fluid. For the remaining histochemical reactions fixation of 24 hr in Bouin, Susa and 10% formalin was utilized. The material was washed, dehydrated, and embedded in paraffin (56°C). Serial sections were cut at 7 pm. The neurosecretory material was stained with Gomori’s chrome hematoxylin-phloxine and PF methods. Azocarmine and Masson’s trichrome methods were also employed. The following histochemical tests were used: (1) the performic acid-Al&n blue (PFAAB) method for disulfide groups, after

Adams and Sloper (1956) ; the test was also carried out replacing the performic acid with the permanganic acid (PMAAB) (cf. Lison, 1960); (2) the dihydro22/-dinaphthyZ-disulfide (DDD) technique for the demonstration of sulfhydryl (-SH) groups, after Barrnett and Seligman (1952) ; (3) the thioglycolate-DDD technique for -SS- and -SH groups (cf. Pearse, 1968) ; (4) the thioglycolate-ferric fcrricyanide method to demonstrate -SS- and -SH groups, according to Adams (1956) ; (5) the thioglycolate-alkaline tetrazolium for -SS- and -SH groups (cf. Pearse, 1968) : (6) the p-dimethylamino benzaG dehyde-nitrite (DMAB-nitrite) method for tryp-

454 Copyright @ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.

NEUROSECRETION IN Enchytraeus 455

tophan and other 3-indolyl derivatives, after

Adams (1957) ; (7) the Millon test for tyrosine (cf. Pearse, 1968); (8) the chtorumine ‘I-Schij method for protein-bound NH,, according to Burstone (1955); (9) the tetrazonium reaction for protein in general (cf. Pearse, 1968) ; (10) the Sakaguchi reaction for arginine (cf. Pearse, 1968) ; (11) the Sudan black B (SBB) staining for lipids in paraffin sections, according to Lison (1960); (12) the periodic acid-Schiff (PAS) technique, after McManus (cf. Pearse, 1968).

RESULTS

The results of this research are sum- marized in Table 1, in which are indicated the responses of the neurosecretory cell types to the various tests.

Staining Reactions

With PF both Q and U cells stained vigorously, while P cell secretion was very weakly positive. With chrome hematoxylin U cell secretion stained vigorously, Q cell secretion stained scarcely, P cell secretion was completely negative. With Azocar- mine and Masson’s trichrome methods, the neurosecretory cells were not demonstrable.

Histochemical Reactions

PFAAB. Both Q and U cells were char- acterized by the strong response of their

secretions to this test. The P cell secretion was weakly positive to the test. A intense response was observed also in the axons of Q cells. This test is used for the demon- stration of disulfide linkages.

PMAAB. The secretions of Q, U, and P cells stained strongly with this test (Fig. 1).

DDD. A weak response was noted with DDD in the cytoplasm of both Q and U cells. The secretions of the P cells were negative to the test. The DDD technique is specific for the demonstration of sulf- hydryl groups.

Thioglycolate-DDD. Here the staining with DDD was preceded by the incuba- tion (37°C) of the sections in sodium thioglycolate adjusted to pH 8. Thiogly- colate reduces disulfide to sulfhydryl. The thioglycolate-DDD technique demonstrates both free sulfhydryl and disulfide groups. With this method the secretion of both Q and U cells stained red (Fig. 2). In the cytoplasm of the P cells the reaction was negative.

Thioglycolate-ferric ferricyanide. With this technique a positive response was noted in the cytoplasm of both Q and U cells indicating the presence of -SS- and -SH

TABLE 1 STAINING REACTION AND HISTOCHEMISTRY OF THE NEUROSECRFXORY M~TERI~TP

Method Q cells u cells P celk Significance

Azocarmine Masson PF Chrome hematoxylin-phloxine PFAAB PMAAB DDD DDD (thioglycolate) Thioglycolate-ferric ferricyanide Thioglycolate-alkaline tetrazolium DMAB-nitrite Millon Chloramine TSchiff Tetrazonium Sakaguchi SBB PAS

- - - -

+++ +++ ++ +++

+++ +++ +++ +++

+ + +++ +++ ++ ++

+++ +++

+++ +++

- -

- + - + Disulfide

++ Disulfide - Sulfhydryl - Sulfhydryl and disulfide - Sulfhydryl and disulfide

+ Sulfhydryl and disulfide - Tryptophan - Tyrosine - Protein-bound NH, - Protein - Arginine

+++ Lipid - 1 ,zGlycol

a Symbols: - = negative reaction; +, ++, ++ + = reaction more or less st,rongly positive.

4.56 STEFANO BIANCHI

FIG. I. A Q cell stained with PMAAB. x2200. FIG. 2. A Q cell stained with thioglycolate-DDD. x 1400.

FIG. 3. .4 Q cell stained with thioglycolate-alkaline tetrazolium. X 1600. FIG. 4. A P cell stained with SBB. x2000.

groups. The P cells did not stain with this method.

Thioglycolate-alkaline tetrazolium. The Q and U cell secretions showed a strong reaction to this test (Fig. 3). In the cyto- plasm of the P cells the response was faintly positive.

DMAB-nitrite. The DMAB-nitrite tech- nique gave a negative response in all neu- rosecretory cells types.

Millon. This technique is employed for demonstrating the presence of proteins containing tyrosine. The Q, U, and P cell secretions showed negative reactions with this technique.

Chloramine T-Schif. This technique is recommended as general protein stain. The secretions of the Q and U cells showed a strong staining reaction with this tech- nique. The reaction in the P cells was negative.

Tetrazonium. The neurosecretory cells did not stain with this method, which is employed for protein in general.

Sakaguchi. The Sakaguchi reaction for arginine was negative in all neurosecretory cell types.

XBB. The secretion of P cells showed a strong staining with the SBB technique for lipids (Fig. 4). The secretions of both Q and U cells did not stain with this method.

PAS. This technique was employed as a test for the presence of carbohydrates. The secretions of Q, U, and P cells were nega- tive to the test.

DISCUSSION

From the range of histochemical reac- tions employed it appears that the secre- tions of both Q and U cell types contain disulfide groups. There was no indication in the secretory product of these cells of the presence of carbohydrate, lipid, and tryptophan. Disulfide groups were demon- strated by thioglycolate-DDD, thiogly- colate-ferric ferricyanide, thioglycolate- alkaline tetrazolium, PFAAB and PMAAB tests. These groups may be derived from

NEUROSECRETION IN Enchytmeus 457

cystine (Barrnett, 1954; Barrnett and Seligman, 1954 ; Sloper, 1954)) which is a principal component of the peptide neuro- secretion in invertebrates and vertebrates. The histochemical tests employed for pro- tein were negative with the exception of the chloramine T-Schiff technique. With reference to the Sakaguchi and Millon reactions, it is possible that neither arginine nor tyrosine is present in the secretions of both Q and U cells. The negative results with tetrazonium reaction remain unex- plained.

In contrast to the Q and U cell secre- tions the P cell secretion has not a pro- teinaceous character. The P cells, which did not take chrome hematoxylin and stained slightly with PF, gave a completely negative response to the major histochem- ical tests to which the Q and U cells showed a positive reaction and to all of the other techniques applied for protein. The P cell secretion was observed very well in the sections stained with SBB tech- nique for lipids. The presence of lipid com- pounds in the P cells appears indisputable and in this the observation of Deuse- Zimmermann (1960) agree with our own.

The histochemical investigations on neurosecretion in Oligochaeta have been carried out on Lumbricidae. In the earth- worm Octolasium complanatum neurosecre- tory cells whose secretions are rich in disulfide groups have been described (Bianchi, 1963a,b). In this respect these cells are comparable to the Q and U cells of Enchytraeus. In Octolasium neurosecre- tory cells comparable with the P cells of Enchytraeus are absent. In Eisenia foetida neurosecretory cells whose secretions con- tain a PAS-positive substance have been described (Herlant-Meewis, 1955).

Studies on the histochemistry of neuro- secretion have been carried out in other an- nelid classes (Hirudinea and Polychaeta) , in nemerteans, in insects and crustaceans. In the Hirudinea the secretions of the neurosecretory cells are found to be pro- teinaceous with possible small carbohy- drate moieties (Bianchi, 1964; Hagadorn, 1966) and lipid moieties (Hagadorn, 1966). Some of these neurosecretory cells (p2 cells), have a high content of tryptophan

or other 3-indolyl derivatives (Hagadorn, 1966). In the Polychaeta a PAS-positive component has been observed in some of the neurosecretory cells (Arvy, 1954; Gabe, 1954; Defretin, 1955; Clark, 1955). A lipid component was reported in neurosecretory cells of certain Polychaeta (Bobin and Durchon, 1952; Clark, 1955). In Cerebra- tulus marginatus (Heteronemertini) the neurosecretory cells (a, b, and c cells) are characterized by a content of cystine and tryptophan or other 3-indolyl derivatives (Bianchi, 1969). The presence of disulfide groups has been reported in neurosecretory cells of insects (Sloper, 1957; Arvy and Gabe, 1961; Pipa, 1961) and crustaceans (Rehm, 1959). Furthermore there are data on a lipid component and a PAS-positive component in neurosecretory cells of in- sects and crustaceans (see Gabe, 1966).

The finding in Enchytraeus of a neuro- secretion rich in disulfide groups extends to the lower Oligochaetes the observations that a cvstine-rich component is character- istic of certain neurosecretions of the Lumbricidae, Hirudinea, Heteronemertini, arthropods (see above) and of the verte- brates (e.g., Barrnett and Seligman, 1954). It is interesting to note that in Enchy- traeus, as well as in the above-mentioned invertebrates and in the vertebrates, the neurosecretions rich in cystine are also the ones which stain with PF.

Another notable fact is that the P cells of Enchytraeus stain strongly with the SBB technique for lipids. It seems that the secretory material of these cells is com- posed largelv, if not solely, of lipids. This is a peculiarity of the Enchytraeidae among the Annelida. In fact in the Poly- chaeta the nellrosecretorv material con- tains only a lipid moiety (Bobin and Dur- chon, 1952; Clark, 1955)) in the Hirudinea the secretions of the neurosecretory cells ((Y, pz, 6 cells) show a weak to moderate staining with SBB (Hagadorn, 1966) and in the Lumbricidae the neurosecretory ma- terial does not appear to contain a lipid component (Bianchi, 1963a,b).

ACKNOWLEDGMENTS

I am grateful to Professor Baldasaarre de Lerma for helpful advice and criticism. The pres-

45s STEFAXO BIAK’CHI

ent work was cairicd out with the finamial aid of the Consiglio Nazionale delle Ricerche of Italy. I wish to thank Mr. Salvatore Cioffi for technical assistance.

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