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May 16, 1977 - Van Orden, L. S., III (1976) in SIF Cells:Structure andFunction .... 44. von Euler, U. S. (1956) Noradrenaline (Thomas, Springfield,. IL). 45.
Proc. Nati. Acad. Sci. USA

Vol. 74, No. 8, pp. 3587-3591, August 1977 Neurobiology

Occurrence of somatostatin-like immunoreactivity in some peripheral sympathetic noradrenergic neurons lamine precursor uptake and decarboxylation (A.PUD)-concept/Dale's principle/peripheral peptide neurons] T. HOKFELT*t, L. G. ELFVINf, R. ELDE*§, M. SCHULTZBERG*, M. GOLDSTEIN$, AND R. LUFT'1 Departments of *Histology and tAnatomy, Karolinska Institute, I1Department of Endocrinology, Karolinska Hospital, Stockholm, Sweden; and IDepartment of

Psychiatry, New York University Medical Center, New York, New York 10016 Contributed by Rolf Luft, May 16, 1977 ABSTRACT By means of the indirect immunofluorescence technique of Coons and collaborators, somatostatin-like immunoreactivity has been demonstrated in principal ganglion cells of some sympathetic ganglia. The noradrenergic nature of these cells was established by "staining" of the same or consecutive sections with antiserum to dopamine P-hydroxylase

Idopamine f-monooxygenase; 3,4-dihydroxyphenylethylamine,

ascorbate:oxygen oxidoreductase (hB-hydroxylating), EC 1.14.17.1J, the enzyme converting dopamine to noradrenaline (norepinephrine). In guinea pigs the somatostatin immunoreactive material was found in almost two-thirds of all principal ganglion cells of the coeliac-superior mesenteric ganglion complex (anterior inferior part) and of the inferior mesenteric ganglion, but only in a few cells of the superior cervical ganglion. It appeared to be localized close to the Golgi complex. The present findings may represent a concomitant storage of a biogenic amine and a imall-peptide in a neuron. Because both noradrenaline and somatostatin may fulfill a role as a neurotransmitter or modulator, the sympathetic neurons described in this study may represent an example of mammalian nerve cells not conforming to Dale's hypothesis, i.e., the one neuronone transmitter concept. In some peripheral tissues, notably the gastrointestinal tract, cells of endocrine nature exist which appear to store and/or to synthesize concomitantly a peptide hormone and a biogenic amine (see refs. 1-3). Pearse (1) considers these cells as a particular system and has termed them "APUD cells" (amine content and/or amine precursor uptake and decarboxylation). They appear to have a common origin from the neuroectoderm of the neural crest (1). So far, however, no conclusive evidence has been presented for the case of a neuron belonging to the APUD system, although this possibility has been advanced by Pearse (4). Such a hypothetical neuron would be of particular interest because monoamines as well as some small peptidesamong others angiotensin II (5), substance P (6, 7), thyrotropin releasing hormone (TRH) (8, 9), and somatostatin (9)-may act as neurotransmitters or neuromodulators. Such a finding would not conform with Dale's principle, according to which each nerve cell makes and releases only one transmitter. The coexistence of more than one putative transmitter in a neuron has, however, been demonstrated in invertebrate neurons (see, e.g., refs. 10-12) and the validity of Dale's principle has recently been discussed by Burnstock (13). In the present article evidence is presented for the existence of somatostatin or a somatostatin-like peptide in some peripheral adrenergic neurons of mammals. The evidence is based on indirect immunofluorescence microscopy with antisera to somatostatin and dopamine fl-hydroxylase [DBH; dopamine f3-monooxygenase; 3,4-dihy-

droxyphenylethylamine,ascorbate:oxygen oxidoreductase (0-hydroxylating), EC 1.14.17.1]. This enzyme converts dopamine to noradrenaline (norepinephrine) and serves as a marker for noradrenaline (and adrenaline) neurons (see, e.g., refs. 14-19). MATERIAL AND METHODS Antiserum to synthetic somatostatin (Kabi, Stockholm) conjugated to keyhole limpet hemocyanin (KLH) was raised in rabbits as described elsewhere (20). In the present study bleeding R 141 C was used. DBH was purified from cow adrenal glands and antiserum to the enzyme was raised in rabbits. These procedures have been described in detail elsewhere (21). Male guinea pigs (body weight 250-350 g) and male albino rats (Sprague-Dawley, body weight 150 g) were used. The animals were perfused via the ascending aorta with ice-cold 4% formaldehyde in 0.1 M phosphate buffer prepared according to Pease (22). After perfusion for 30 min the following ganglia of the guinea pig were dissected out: the superior cervical ganglion, the coeliac-superior mesenteric ganglion complex, and the inferior mesenteric ganglion. The coeliac-superior meserteric ganglion complex was divided into an anterior inferior and a posterior superior part. In addition, some adjacent ganglionic tissue along the most proximal part of the abdominal aorta was exstirpated. In the rat the same ganglia and the middle cervical ganglion were- studied. Following further 90-min immersion fixation (as above) the tissues were rinsed in 0.1 M sodium phosphate buffer with 5% sucrose for at least 24 hr, cut in 10-,um thick sections on a cryostat (Dittes, Heidelberg), and processed for indirect immunofluorescence microscopy principally according to Coons and collaborators (see ref. 23). Briefly, series of four consecutive sections were incubated in a humid atmosphere at 370 for 30 min with antisera to somatostatin (dilution 1:20) or DBH (dilution 1:20) or control sera. After rinsing in phosphate-buffered saline the sections were incubated under the same conditions as above in fluorescein isothiocyanate-conjugated sheep antiserum to rabbit 'y-globulin (dilution 1:4) (Statens Bakteriologiska Laboratorium, SBL, Stockholm, Sweden), rinsed in phosphate-buffered saline, mounted in glycerol/phosphate-buffered saline (3:1), and examined in a Zeiss fluorescence microscope. Somatostatin antiserum blocked with an excess of somatostatin (antiserum at 50 ,qg/ml diluted 1:10) and normal rabbit serum served as control sera. All sera contained 0.3% (vol/vol) Triton X-100 (16). Abbreviations: DBH, dopamine fl-hydroxylase; APUD, amine precells. t To whom reprint requests should be addressed. § Present address: Department of Anatomy, University of Minnesota, Minneapolis, MN 55455.

The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

cursor uptake and decarboxylation; SIF cells, small intensely fluorescent

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I

FIG.. 1. (Legend appears at bottom of the following page.)

Proc. Natl. Acad. Sca. USA 74 (1977)

Neurobiology: H6kfelt et al. In some cases sections incubated with somatostatn (i; fluorescein-conjugated) antiserum were photographed andie`incubated with DBH (and fluorescein-conjugated) antiserum and rephotographed. The reversed procedure was also carried out, i.e., incubation with DBH antiserum followed by somatostatin antiserum. To obtain a rough estimate of the occurrence of somatostatin immunoreactive neurons, the somatostatin-positive cell body

profiles were counted on fluorescence micrographs (magnification X180) of representative areas of sections incubated with somatostatin antiserum. The sections were subsequently reincubated with DBH antiserum and the same areas were rephotographed. All principal ganglion cell profiles, i.e., DBHpositive as well as DBH-negative cell profiles, were counted. Thus, the percentage somatostatin-postive cells obtained refers to the total number of principal ganglion cells. No attempt has been made to correct for the fact that the volume of the somatostatin-reactive material, which is probably bound to the Golgi apparatus, is smaller than the cell body volume. The

figures obtained are therefore only approximate. RESULTS In the guinea pig somatos~tatin-like immunoreactivity was observed in certain principal neuronal cell bodies'in all sympathetic ganglia studied (]Fig. 1'A, C, D, E, H, and I). No evidence for a localization in small intensely fluorescent (SIF) cells (see ref. 24) could be obtained (Fig. 1A). The immunofluorescence was localized exclusively to the cytoplasm. It was not diffusely distributed but had a coarse granular appearance, often forming a continuous perinuclear ring and sometimes extending for a short distance into a cell process (Fig. 1H). The number of immunoreactive cells differed widely among the ganglia studied. The inferior mesenteric ganglion (Fig. 1A) and the anterior inferior part of the coeliac-superior mesenteric ganglion complex (Fig. 1E) contained the highest concentrations of somatostatin-positive cells, with almost two-thirds of all principal ganglion cells labeled. In the former ganglion 286 out of 458 (62.5%) cells were labeled as compared to 2365 out of 3990 (59.3%) in the latter. The positive cells were comparatively evenly distributed over the sections, but groups of cells often peripherally located could be completely devoid of somatostatin-like immunoreactivity. In the remaining ganglia considerably fewer imm unoreactive cell soma were observed. In the posterior superior part of the coeliac-superior mesenteric ganglion complex about 25% (91 out of 342 cells) and in ganglia along the aorta only about 7% (33 out of 459 cells) were labeled. In the superior cervical ganglion only single somatostatinpositive cells were seen (Fig. 1 C and D). In addition to cell bodies, varicose somatostatin-immunoreactive fibers were also observed. Incubation of adjacent sections with antiserum to DBH (Fig. 1 B and F) revealed that the vast majority of' the somatostatin-positive cells also were DBH-positive, i.e., represented nor-

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adrenergic ganglion cells. This could also be established by incubations of the same section with both anti-

consecutive sera.

In the rat, ganglion cells in the inferior mesenteric ganglion, the coeliac-superior mesenteric ganglion complex, and the middle cervical ganglion contained a weak somatostatinimmunoreactive material with an appearance-and distribution similar on the whole to that found in the guinea pig principal ganglion cells. No attempts to quantify these cells were made, but they were less numerous than in the guinea pig. Incubation of consecutive sections with DBH antiserum strongly suggested that the somatostatin-positive cells are catecholamine neurons.

No immunofluorescence was observed in the sympathetic ganglia after incubation with control sera.

DISCUSSION The present findings indicate that certain neuronal cell bodies in some sympathetic ganglia of the guinea pig and to some extent of the rat contain both the enzyme DBH'and somatostatin or a somatostatin-like peptide. DBH converts dopamine to noradr9.naline and is an accepted marker for noradrenergic neurons (see, e.g., refs. 14-19) and, in the central nervous system, for adrenergic neurons (see ref,17). The poradrenergic nature of the vast majority of the ganglion cflls in the superior cervical ganglion, the inferior mesenteric, and coeliac ganglia has previously been demonstrated both with formaldehydeinduced fluorescence (see refs. 25-30) and immunohistochemistry (14, 15, 17-19, 31) and was confirmed in this study. Somnatostatin was isolated and characterized as a tetradeca' peptide by Brazeau et al. (32). It represents the growth hormone release-inhibiting factor discovered by Krulich et al. (33). Moore recently it has, however, become apparent that somatostatin has a wide distribution in the central nervous system (34-38). In the peripheral nervous system somatostatin has up till now been described only in primary sensory neurons and in nerve fibers in the intestinal wall (34, 39). The present results suggest that certain noradrenergic sympathetic neurons, especially in some prevertebral ganglia, may also synthesize and store this peptide. It is important to underline that this appears to be true only for part of the noradrenergic neurons, because somatostatin-like immunoreactivity is missing in several DBH-positive neurons.

So far somatostatin has been identified with certainty only in the perikarya pf the noradrenergic neurons. The exact subcellular storage site for somatostatin in these cells is difficult to define on the basis of light microscopy. The staining pattern observed is, however, remarkably similar to that seen 'with stainings assumed to specifically visualize the Golgi apparatus of sympathetic neurons (40). In fact, localization of somatostatin to the Golgi complex has also been observed in specific populations of primary sensory neurons and in central neurons (41).

FIG. 1. A-I (on preceding page). Immunofluorescence micrographs of the inferior mesenteric ganglion (A, B, and I), the superior cervical ganglion (C and D), and the coeliac-superior mesenteric ganglion complex (the anterior inferior part, E-H) after incubation with somatostatin antiserum (A, C-E, and H), with dopamine fl-hydroxylase antiserum (B and F), with both somatostatin and dopamine fl-hydroxylase antiserum (I) or with control serum (somatostatin antiserum blocked with excess of somatostatin) (G). A and B on one hand and E-G on the other hand show consecutive sections. In the inferior mesenteric ganglion (A) as well as in the anterior part of the coeliac-superior mesenteric ganglion complex (E), the majority of the principle ganglion cells are somatostatin-positive, whereas the small intensely fluorescent (SIF) cells (asterisks) lack a specific fluorescence (A). The superior cervical ganglion contains only a few positive cells (C and D; D represents a higher magnification of C). The somatostatin-immunoreactive material appears to be localized to the Golgi apparatus (H). Most of the principle ganglion cells as well as the SIF cells contain dopamine ,B-hydroxylase (B, F, and I), indicating that they represent catecholamine neurons. After consecutive incubations with both antisera (I) the fluorescence is localized both to the Golgi apparatus and to a pool in the cytoplasm. There are also many fluorescent, mainly dopamine-fl-hydroxylase-positive nerve endings in the inferior mesenteric ganglion (I). Note lack of immunoreaction after incubation with control serum (G). Magnifications X120 (A-C and E-G) and X300 (D, H, and I).

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Sometimes, however, a more diffuse cytoplasmic staining is seen (see, e.g., ref. 39). The reason(s) for this variability are not completely known but a more diffuse staining could either indicate diffusion of somatostatin out from the Golgi complex due to poor fixation, or, more likely, the presence also of cytoplasmic somatostatin obscuring the Golgi-related pool, in this case indicating an especially efficient fixation. An artefactual association of somatostatin to Golgi membranes cannot be completely ruled out. Whether or not the somatostatin-immunoreactive material is confined to the cell soma cannot be decided at the moment. Our studies on the distal colon, a part of then gut known to be innervated by the inferior mesenteric ganglion, demonstrate both DBH-positive (noradrenergic) and somatostatin-positive fibers in the myenteric (Auerbach's) plexus, but an identity cannot be established at the light microscopic level (unpublished). It is interesting to note that somatostatin-like immunoreactivity has been described in the pineal gland (42, 43). Whether or not it is present in noradrenergic nerve terminals originating in the superior cervical ganglion is not known. The present findings give evidence that not only endocrine-like cells (see refs. 1-3) but also a neuron can concomitantly store a biogenic amine and a biologically active peptide, extending the APUD concept of Pearse (1). Whether or not other monoamine neurons also contain somatostatin or some other biologically active peptide is at present unclear. We have applied the same technique as in this study to the central nervous system using antisera to somatostatin and other peptides and to catecholamine-synthesizing enzymes, but in no case has there been a conclusive demonstration of a concomitant storage (38). Thus, in the anterior periventricular hypothalamic region both dopamine and somatostatin cells are found, but analysis with consecutive incubations could not establish identity (38). It must be emphasized that these preliminary findings do not exclude cases of amines and peptides being present in the same central neurons. With regard to functional role there is little doubt that noradrenaline, in both the peripheral and the central nervous system, is a transmitter substance (44-46). Somatostatin, on the other hand, is less well characterized, but in several endocrine cell systems it appears to exert a powerful inhibitory action on secretion of many hormones (see, e.g., refs. 35 and 47). Also in the central nervous system somatostatin has been shown, by means of neurophysiological techniques, to depress neuronal activity (9), possibly via an effect on Ca2+ transport (48). The possibility should be considered that somatostatin, like other peptides such as angiotensin 11 (5), substance P (6, 7), and thyrotropin releasing hormone (8, 9), may be either a neurotransmitter or a neuromodulator. Should this turn out to be true, our findings would present a possible case for the existence of two transmitters in one mammalian neuron. This hypothesis must, however, be strengthened by the demonstration of somatostatin not only in noradrenergic perikarya but also in noradrenergic nerve terminals, i.e., at the sites of release. As discussed above this has so far not been achieved. The fact that some invertebrate neurons appear to contain several putative transmitters (10-12), and other reasons, has led Burnstock (13) to suggest a reexamination of the "one neuron-one transmitter" concept of Dale. Our findings support this suggestion. The present findings underline the view that peptides may play important roles not only in the central but also in the peripheral nervous system. In the intestinal wall neuronal stores of immunoreactive substance P (49, 50), somatostatin (34), vasoactive intestinal polypeptide (VIP) (51-53), enkephalin (54), and angiotensin II (55) have been found. Furthermore,

in the coeliac and inferior mesenteric ganglia dense networks of substance P- (56), enkephalin- (57), and VIP-positive nerve terminals and VIP-positive cell bodies (58) are present. The origin and function of these peptide systems are not known. In a review article Hillarp (59) concluded from the experiments of, among others, Kuntz and collaborators (see ref. 60) that it seems "necessary to postulate the existence of reflex systems involving synaptically connected neurons wholly confined to the peripheral autonomic nervous system." Perhaps at least some of the peptide neurons just mentioned are involved in such reflexes. It is noteworthy that both substance P (39, 61) and somatostatin (39) appears to be present in spinal ganglion cells and thus possibly take part in the reflex mechanisms mediated via primary sensory neurons. The skillful technical assistance of Miss A. NygArds, Mrs. W. H. Hiort, Mrs. Lena Hammarberg, and Miss Britt Dahlgren is gratefully acknowledged. This work was supported by gr~ants from the Swedish Medical Research Council (04X-2887, 19X-3412), Magnus Bergwalls Stiftelse, Harald och Greta Jeanssons Stiftelsp, and funds from the

Karolinskii Institutet. Somatostatin was supplied by Kabi Research Laboratories, Stockholm. R.P.E. is a recipient of a National Institutes of Health National Research Award (NS 0547-01) from the U.S. National Institute of Neurological and Communicative Disorders and Stroke. 1. Pearse, A. G. E. (1969) J. Histochem. Cytochem. 17,303-313. 2. Owman, Ch., HAkansson, R. & Sundler, F. (1973) Fed. Proc. 32,

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