Neuroscience Le~ters, 10 (1978) 187--191 © Elsevier/North-Holland Scientific Publishers Ltd.

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I M M U N O F L U O ~ N T LOCALIZATION OF DOPAMINE~.HYDROXY- LASE IN SMALL INTENSELY FLUORESCEI~r CELLS OF THE RAT SUPERIORCERVICAL GANGLION

RAINER K~)NIG and CHRISTINE HEYM

Anatomisches Institut der Universitlit D-6900 tleidelb~rg, lm Neuenheimer Feld 307 (G.F.R.)

, (Received July 24th, 1978) (Accepted July 24th, 1978)

SUMMARY

Serial sections of the superior cervical ganglion of n~wbom and adult rats have been treated alternatively with fluorescent labelled antibodies to dop- amine~-hydroxylase (DBH), or with glyoxylic acid. Two different cell types could t ~. distinguished: (1) a small intensely fluorescent (SIF)-cell majority demonstrating a bright ~lyoxylic acid-induced fluorescence but no DBH-positive fluorescence; (2) a minor cell population bring flum~scent following both treatr~ents. The findings indicate the presence of norepinephrine (and/or epinephrine)-containing SIF-cells in the r~Lt superior cervical gangliou.

Apart from the principal neurons, small paraganglionic cells in sympathetic ganglia have been found to exhibit an intense yellow-green paraformah4.ehyde, or glyoxylic acid, induced fluorescence (for review see ref. 3) indicaJ mg a high content of monosmines. Therefore these cells are called small intensely fluores- cent (SIF) cells [5].

Whereas the histochemical evidence that the intracellular moz,oamines belong to the catecholamine-group has become well accepted [ 13], '~e nature and identity of the catecho|emine have been the subject of controversy.

Micr0spectro-fluorometric investigations have indicated that SIF-cells in the rat superi, or cervi'cal gangUoncontain most of the dopamine (DA) pre~nt in the g~nglio n [2]. From theabsence of the enzyme dopamine-~-hydroxylase (DBH)Im SIF.cells, as demonstrated by immunohisto,~emical methods, it was concluded: that ~ ¢ ~ contain no norepinephrine (N E, see ref. 6). However, t~le d i f f ~ t ~ r e s t : O f 8iF-cells to formaldehyde vapour [ 14] or to glutaraldehyde dichromate tnmtment [14], as w~l~ as th~ ultrastructur~d divers- ity of catecholamine-containing granules within different paraganglioni0: cells led to the sssumption of two SIF-ceU populations [8,12] one of whi,:h is supposed to c~ntain NE [11].

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Considering these contradictory data the purpose of this investigation was to discriminate between DA-containing and NE-containing SIF-cells in the rat superior cervical ganglion by means of the immunofluorescent demonstration of intracellular DBH, the enzyme involved in the conversion of DA to NE.

8 adult male Wistar rats (body weight 150--200 g) and 5 new-born animals (3 days postnatally) of the same strain were killed by exsanguination. The superior cervical ganglia were rapidly dissected out and frozen in Isopentane which has been cooled by liquid nitrogen. Alternating 12 ~m cryostat serial sections were prepared either for FITC-labeUed immemofluorescence CA), or for glyoxylic acid-induced fluorescence (B). A: Section~ were fixed for 20 rain in phosphate-buffered 4% parafonnaldehyde and washed in 0.1 M phosphate buffer for at least 30 rain according to the description of H6kfelt et al.[9]. : The sections were then processed for the indirect immunofluorescence tech- nique as described by Hartmann [7]. Antisera to DBH were diluted I : 10, the solution containing 0.3% Triton X-100 (Sigma Lab.). The incubation were performed at 37°C for 15 rain. The sections were incubated with fluor~scein isothiocyanate (FITC)-conjugated goat antirabbit antibodies (Miles Lab.) under the same conditions described above. B: A modified glyoxylic acid-induced fluorescence method [3] was applied to the other half of alternating serial sections for visualization of dopaminergic SIF-cells.

All sections were examined with a Zeiss epiilluminator condensor III RS, using the filters BG :12, FT_ 510, 50.

Groups of small SIF-cells exhibited a bright yellow-green glyoxylic acid- induced fluorescence (Fig. 2a). These groups were scattered between weakly fluorescent principal neurons in the superior cervical ganglion of newborn as well as of adult animals; they were usually clustered near blood capillaries.

a ! " ' b

o

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•

Fig. 1. Strongly DBH-positive cell clusters in close proximity of capillaries (dark lummina) in superior cervical ganglia. (a) adult; (b)three days postnatally.

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Fig. 2. CorresponJing areas in two consecutive sections of the superior cervical ganglion of an adult rat. Numbers indicate identical principal neurons in transverse sections. (a) Glyoxylic acid-induced fluorescence reveals the presence of two clusters of SIF-cells among weakly fluorescent principal neurons; (b) No DBH-positive immunofluorescence is observed in the upper right SIF-celi cluster (dark region between DBH-positive l~rincipal neurons 1, 2 and 3 ~ Three small weakly DBH-positive cells are located at the site of the IG-,~,er left SIF-cell cluster (arrow).

Besides moderately DBH-positive principal neurons, a minor proport ion of SIF-eells exhibited a DBH-positive fluorescence of varying intensity. Some of these cell groups were distinctly brighter than the surrounding principal neurons (Fig. l a , b), indicating a high DBH-content. Other SIF-cells, weakly DBH-positive, could only be distinguished from principal neurons by their size and by their location at the site of an intensely fluorescent SIF-cell cluster seen in adjacent glyoxylic acid-treated sections (Fig. 2a, b).

Sometimes only part of a glyoxylic acid-induced fluorescent cell cluster was also DBH-positive (Fig. 2b); in other cases, all small cells of one cluster were both glyoxylic acid-induced fluorescent and DBH-positive.

In all cases, DBH-positive SIF-cells in adult were clustered into groups of 3 to 20 cells (Fig. la ) while in newborn animals, the groups consisted of 2 to a maximal 14 cells (Fig. lb) .

DBH-positive SIF-cell groups were found at random locations, showing no preference to ganglion pole. They amounted to 5--10% of the total SIF-cell number/gang!.~on. No singly located DBH-posithre SIF-cells were seen in the ganglia of either newborn or adult rats.

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In 5 out o f 1 6 adult ~ : ~ , ) B H , p o s i t i v e 8IF~e!!S were not found at all; only in I m sl~ Were!~ both ~ p , liafound to!ackDBH:positive SlF'ceUs. The present Study Shows tha t the p ~ c e o f DBHcan be proved in a minor SIF- cell populat lonof the rat sUperior i ~ a l g a n s i i 0 i ~ indicating ~that the amine in these cells is NE i The identification holds bothYor newbom and for adult animals. " ' ~... . . . . .

The findings confims~those of Lever et al. [11] who demonstrated the pre- sence of NE by c'h~omitlm positive granules in a small SIF~ell population of the rat superior ce rv ica /~gJ ion using X-ray microanalysis after glutaraldehyde d~-~romate treatment. ~ •

• . . . . .

The present data are als0 in accordance With the ultrastructural observation of a paraganglionic cell type characterized by catecholamine storage granules resembling those in NE.cells of the adrenal medulla [12].

Cur results, however, are in c( ntrast to reports concluding the superior cervical ganglion SIF~elis not to contain DBH: thus being purely dopaminergic [1,6,7].

The DBH content of SIF~ells appears to vary considerably. Only a very small number of SIFCells stain strongly DBH-positive, these not being found in every ganglion. This could explain the lack of positive immunofluorescent SIF-ceUs noted by other investigators [1,6,7] as well as the absence observed in some ganglia in our study. Furthermore, the immunofluomscent reaction to DBH seen in principal neurons may have masked the fluorescence of weakly DBH-posit~ve SIF-cells, as demonstrated in Fig. 2b (arrow). In that case, neg- ative results may have merely indicated that DBH was present in amounts too low to be visualized by the applied technique.

It remains to be elucidated whether the same cell contains mote than one catechol~mine in a form available for release [4]. It is possible for instance that DA is also present in those structures which am characteristic of NE- containing ceils.

Speculating on the functions~ implication of SIF-cells, only DA-ceUs were shown to be interneuronally co mected [12], whereas NF~ells have been observed in close proximity to regional capillaries within sympathetic ganglia [4 ]. These observations are in accordance with our findings that D BH-positive cells are always clustered near blood vessels. This close vascular proximity makes a neurosecretory function of NE-cells conceivable [14].

ACKNOWLEDGEMENTS

This work was supported by the Deutsche Forschungqem~:inschaft grant He 919/3.

We wish tc thank Dr. G. Feurle, Medizinische Poliklinik H~:idelberg, for his valuablehelp in raising antibodies to DBH.

Our thmlks are slso due Prof. M. Goldstein, Dept. NeurochL,~n., N.Y. Medical Center, New York 10016 for the kind disposition of control l;'BH-antisera and to Dr. G. Taugner, Max-Planck-lnstitut f. Med~Forschung, Heidelberg, for the donation of purifiect DBH.

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1 Baker, H.A., Burke, J.P., Bhatnangar, R.K., Van Orden, D., Van Orden L. and Hartman, B.K., Histochemical and biochemical characterization of the rat paracervical ganglion, Brain Res., 132 (1977) 393--405.

2 Bj6rklund, A., Cagrell, L., Falck, B., Ritz~n, M. and Rosengren, E., Dopamine~ontain- in8 cells in sympathetic ganglia, Acta physiol, scand., 78 (1970) 334--338.

3 De la Torre, J.C. and Surgeon, J.W.~ a methodological approach to rapid and sensitive monoamine histofluorescenee using a modified glyoxylic acid technique: the SPG method, Histochemistry, 49 (1976) 81--93.

4 Elfvin, L.G., H~kfelt, T. and Goldstein M., Fluorescence micro~:opical, immunohisto- chemical and ultrastructurai studies on sympathetic ganglia of the guinea pig, with special reference to the SIF~eils and their catechoI~,r..ine content, J. Ultrastruct. Res., 51 (1975) 377-396.

5 Er~nk~, O. and I-I~rk~nen, M., Monoamine containing small cells in the superior cervical ganglion of the rat and an organ composed of ~.hem, Acta physiol, scand., 63 (1965) 511--512.

6 Fuxe, K., Goldstein, M., H~kfelt, T. and Joh.T.H., Immunohistochemical ,~oalization of dopamine-~-hydroxylase in the peripheral and central noradret, ~rgic nervous system, J. Histochem. Cytochem., 21 (1973) 312--331.

'/ Hartman,. B.K., Immunofluorescence of dopamine-0-hydroxylase. Application of i~- proved methodology to the localization of the peripheral and central noradrenergic nervous system, J. Histochem. Cytochem., 21 (19'/3) 321--331.

8 H:ym,Chr., Einfltum yon Disulfiram auf das Ganglion cervicale superius der Ratte, Verb. Anat. Ges., 70 (1976) 437-445.

9 H~kfelt, T., Fuxe, K., Goldstein, M. and Joh,T.H., Immunohistochemical localization of three catecholamine sythesizing enzymes: Aspects on methodology, Histochem., 33 (1973) 231--254.

10 Kawamura, A., Jr., Fluorescent antibody techniques and their application. Tokyo: University of Tokyo Press and Baltimore, Manchester, University Park Press, 1969.

11 Lever, J.D., Santer, R.M., Lu, K.S. and Presley, R., Electron probe x-ray microanalysis of small granulated cells in rat sympathetic ganglia after sequential aldehyde and dichro- mpte treatment, J. Histochem. Cytochem., 25 (1977) 275--279.

12 Lu,K.S., Lever, J.D., Santer, R.M. and Presley, R., Small granulated cell types in rat superior cervical and coeliac-mesenteric ganglia, Cell Tiss. Res., 1"/2 (19"/6) 331--343.

13 Norbe'rg, K.A., Ritz6v, M. and Ungerstedt, U., Histochemical studies on a special catecholam/ne~ontaining cell type in sympathetic ganglia, Acta physiol, scand., 6"/ (1966) 260--270.

14 Santer, R.M., Lu, K.S., Lever, J.D. and Presley, R., A study of the distributio,~ of chromaffln-poaitive (CH +) and small intensely fluorescent (SIF) cells in sympathetic ganglia of the rat at various ages, J. Anat., (Lond.) 119 (19'/5) 589--599.