GENERAL AND COMPARATIVE ENDOCRINOLOGY 22,24&249 (1974)

The Histochemistry of the Biogenic Monoamines in the Central

Nervous System of Hirudo medicinalis

STEFANO BIANCHI

Institute of Zoology of the University of Naples

Received June 19, 1973

The histochemistry of the biogenic monoamines in the central nervous system of Hirudo is examined. The yellow fluorescent secretion of the small yellow cells is found to be most probably 5-HT. Concerning the large yellow cells, the results seem to indicate an absence of 5-HT. The fluorescent secretion of the green cells is not stainable with the histochemical methods adopted.

The biogenic monoamines have been demonstrated in the neuronal groups of the central nervous system of the medicinal leech by fluorescence microscopy (Bianchi, 1962a) and by the sensitive fluorescence microscope technique of Falck and Hillarp (Kerkut et al., 1967; Coggeshall et al., 1968; Ehinger et al., 1968).

In the present research the biogenic monoamines are studied employing, close to the fluorescence microscopy, a series of histochemical reactions.

MATERIALS AND METHODS

Numerous medical leeches (Hirudo medicinal&) were used in the study. The anterior body seg- ments including the cerebral, subesophageal and ventral ganglia were fixed in 10% formalin for 24 hr, washed, dehydrated, and embedded in paraffin (56°C). The sections were cut at 7 pm, placed on slides according to the ordinary histo- logical technique, and observed with a Wood’s light microscope. A Leitz-Ortholux fluorescence microscope was used. The exciting light of wave- length 365O.k (not strictly mbnochromatic) was provided by a Philips 150-W high-pressure mer- cury lamp with suitable filters. The same sections were treated with the following histochemical methods, which have been considered capable of identifying bodies containing 5-hydroxytryptamine (5-HT) or phenolic compounds (cf. Lison, 1960; Pearse, 1972) : (1) the alkaline diazonium reaction and the acid diazonium reaction (the best results were obtained with freshly diazotized p-nitro- aniline) (cf. Lison, 1960) ; (2) the diazosajranin

method (cf. Lillie, 1965); (3) the argentafin re- action according to Masson-Hamper1 (cf. Lison, 1960); (4) the ch~omafin reaction (the test was carried out on formalin-fixed paraffin-embedded sections) (cf. Lison, 1960); (5) the Gibbs’ 8:6’- diehlo,oquinone-chloroimide method (cf. Pearse, 1972); (6) the alkaline thioindozyl method (cf. Pearse, 1972) ; (7) the Schmorl jeTTic-jerjicyanide method (cf. Pearse, 1972).

RESULTS

The amine-containing neurons of Hirudo may be classified on the basis of their fluorescence in yellow and green cells (Bianchi, 1964, 1967a, 1969; Ehinger et aE., 1968). On the basis of their size, the yellow cells may be distinguished in small and large cells (Fig. 1).

The small yellow cells emit an intense yellow fluorescence. They lie in the ventral nerve ganglia and may be seen also in the subesophageal ganglion. In a ventral ganglion there are four small yellow cells located in the posterolateral groups (Fig. 2). These cells are pyriform in shape whose nucleus is clear with very distinct nucleolus. They are “pseudochromaffin cells” accord- ing to Perez (1943). The fluorescent sub- stance fills most of the cytoplasm. It is confined to the soma of the cell,

The large yellow cells emit a pale yellow fluorescence. They lie likewise the preceding cells in the subesophageal ganglion and in the ventral nerve ganglia. In a ventral

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246 RTEFANO BIASCHI

FIG. 1. Monoamine containing neurons (pointing) of Hirudo. a, green celk; b, small yellow cells; c, large yellow cells.

ganglion there are two large yellow cells located in the anteromedian compartment (Fig. 2). These are the colossal cell of the anteromedian follicle and its neighbor. The large yellow cells are the “true chromaffin cells” of the literature (see Gaskell, 1914; Vialli, 1933; Perez, 1943; Bianchi, 1962a). The fluorescence always appears to be diffused but sometimes areas devoid of fluorescence may be noted in the cytoplasm. The fluorescent substance is confined to the soma of the cell.

/i......., \/ ,/;ch.c.

69 I 1 pch.c.

FIG. 2. Chromaffin cells and colossal cells in a ventral ganglion of Hiwdo. a.m.c., antero-median compartment of ganglion; ch.c., chromaffin cells (large yellow cells); c.c., colossal cells; pch.c., pseudo- chromaffin cells (small yellow celk).

HISTOCHEMISTRY OF THE MONOAMINES IN HIRUDO 247

TABLE 1 COMPARATIVE HISTOCHEMISTRY OF THE MONOAMINE-CONTAINING CELLS OF THE

CENTRAL NERVOUS SYSTEM OF Hirudoa

Fluorescence Small yellow cells

Intense yellow Large yellow cells

Pale yellow Green cells

Intense green

Diazo (alkaline) Diazo (acid) Diazosafranin Argentaffin Chromaffin Gibbs Thioindoxyl Schmorl

+++ t -

+++ f -

+++ - -

+++ +++ -

+ + - - - - - - -

+++ - -

a Range: -, negative; +, weak; +, moderate; ++, strong; +++, very strong.

The green cells emit an intense green fluorescence. They are localized only in the cerebral ganglion. In each compartment of the ganglion there are two or at most three green cells. These cells generally are of small size. The fluorescent substance here occupies all the cytoplasm (Fig. 3).

With the alkaline diazonium test, the fluorescent substance of the small yellow cells was observed to stain orange-red (Fig. 4). In the large yellow cells, the test was weakly positive ; the green cells were negative to the test.

With the acid diazonium test the results were the same observed with the preceding test.

The diazosafranin reaction produces a dark blue staining of the fluorescent sub- stance in the small yellow cells; in the large yellow cells and in the green cells no stain- ing could be demonstrated with this technique.

With the aryentafin test the fluorescent substance of the small yellow cells shows a very strong brown staining; in the large yellow cells the response to the test was also strongly positive (Fig. 5). In the green cells the test was negative.

With the chromafin reaction a moderate

reaction was noted in the cytoplasm of the yellow cells. The green cells were negative to the test.

With the Gibbs method no specific stain- ing could be demonstrated.

The thioindoxyl method did not give a positive response.

With the Schmorl test the fluorescent sub- stance of the small yellow cells appears dark blue. There was no stain in the large yellow cells nor in the green cells (see Table 1).

DISCUSSION

From the intense yellow fluorescence, it appears that the small yellow cells elabo- rate 5-HT. It is known that the tryptamines with formaldehyde are transformed into intensely yellow fluorescent compound (see Falck, 19641. The range of the histo- chemical positive reactions (diazo alkaline, diazo acid, argentaffin, chromaffin, Schmorl) favor our hypothesis that the secretion of the small yellow cells is 5-HT (Bianchi, 196i’al. In this the observations of Berkut et al. (1967) and Ehinger et al. (1968) agree with our own. It is interesting to emphasize here that the reactions described above are the classical histochemical stain-

FIG. 3. Two green cells in a follicle of the cerebral ganglion of Hirudo. Formalin fixing fluid. Photograph in fluorescence microscopy. X700.

FIG. 4. A small yellow cell in the subesophageal ganglion of Hiruch. Note in the cytoplasm the granules stained with diazotized p-nitroaniline. Formalin fixing fluid; alkaline diazonium reaction. X700.

FIG. 5. Two large yellow cells, chromaffin cells (the colossal cell, arrow, and its neighbor), in a ventral ganglion of Hiruclo. Note in the cytoplasm the argentaffin granules. Formalin fixing fluid; aryentaffin reac- tion. X 700.

248 STEFANO BIAKCHI

ing for the enterochromaffin cell granules (cf. Pearse, 1972).

In the large yellow cells the yellow fluorescence seems to indicate the presence of 5-HT, but the partial positive results of the range of the histochemical reactions used-the secretion is clearly demonstrable only with the argent.affin test-do not favor this assumption. This is in disagreement with the observations of Kerkut et al. (1967) and Ehinger et al. (1968)) according to which the large yellow cells probably contain 5-HT.

The green fluorescence of the green cells suggests that these cells contain a catechol- amine, probably norepinephrine. It is known that norepinephrine with formaldehyde forms a green fluorescent product (cf. Falck, 1964). But the intense green fluores- cent material is not demonstrable by means of the histochemical methods adopted.

The small yellow cells of Hirudo are comparable to the yellow fluorescent nerve cells present in the cerebral, subesophageal, and ventral ganglia of the earthworm Octolasium complanatum, whose secretion is stainable with diazotized p-nitroaniline and with other reactions for enterochro- maffin cell granules (Bianchi, 196213, 1963, 1967b). There are not in Octolasium secretory cells comparable to the large yellow cells and green cells of H&do.

Nerve cells stainable with diazotized safranin were observed by Lake (1970) in the nervous system of Chirocephalus diaphanus and by Hinks (1967) in the nervous system of some nocturnal moths. According to the latter, there is in these insects a relationship between the presence of 5-HT and the circadian rhythm.

ACKNOWLEDGMENTS

I am indebted to Professor Baldassarre de Lerma for criticism and advice. This work was carried out with the financial aid of the Consiglio Nazionale delle Ricerche of Italy. I wish to thank Mr. Salvatore Cioffi for technical assistance.

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