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Coexistence of somatostatin- and avian pancreatic polypeptide (APP)-like immunoreactivity in some forebrain neurons
Neuroscience Vol. 1. No. 2. pp. 439 10 446. 1982 Printed m Great Brllain
0306.4522:82/020439-07$03,00/O Pergamon Press Ltd 0 1982 IBRO
COEXISTENCE OF SOMATOSTATINAND AVIAN PANCREATIC POLYPEPTIDE (APP)-LIKE IMMUNOREACTIVITY IN SOME FOREBRAIN NEURONS S. R.
VINCENT*, L. SKIRBOLL*, T. H~KFELT*, 0. JOHANSSON*,J. M. LUNDBERG-~-, R. P. ELDE$, L. TERENIUS#and J. KIMMELT *Departments of Histology and tPharmacology, Karolinska Institutet, Stockholm, Sweden; IDepartment of Anatomy, University of Minnesota, Minneapolis, Minnesota, U.S.A.; $Department IMcllwain
of Pharmacology,
Laboratory,
Uppsala
The University
University.
of Kansas
Medical
Uppsala. Center,
Sweden; Kansas,
U.S.A.
Abstract-The indirect immunofluorescence technique was used to demonstrate the coexistence of somatostatin together with avian pancreatic polypeptide-like immunoreactivity within certain neurons of the rat forebrain. Numerous neurons containing these peptides were observed in the neocortex, hippocampus, olfactory tubercle, striatum, nucleus accumbens and lateral septum. In studies of serial sections stained alternately for these two peptides, and in restaining experiments, it could be determined that in many neurons in these areas these two peptides coexisted. In other brain areas such as the anterior periventricular hypothalamus, somatostatin cells were never found to contain avian pancreatic polypeptide-like immunoreactivity. Also, within the pancreas these two peptides were never found to coexist in the same cells. The findings represent a further example of the coexistence of more than one neuropeptide within a single neuron.
THE GROWTH hormone release-inhibiting hormone, somatostatin, was isolated and structurally characterized in 1973.4 Subsequent immunohistochemical studies demonstrated somatostatin-containing cells in the peripheral and central nervous systems including the gastro-intestinal tracts as well as in pancreas.6 Avian pancreatic polypeptide (APP) was originally isolated from chicken pancreas.” APP-like immunoreactivity in neurons was first demonstrated by Loren, Alumets, Hakanson & SundIe?’ and subsequently by several other groups.12~‘3*22 Both somatostatin and APP have been shown to coexist with either a classical transmitter or with another peptide in the same neuron. Thus, somatostatin-like immunoreactivity has been found in some peripheral sympathetic noradrenergic neurons.’ APP-like immunoreactivity has been shown to coexist with catecholamines in some central and peripheral neurons13.22 and with methionineenkephalin in sacral parasympathetic neurons.i3 Both somatostatin2~7~14~1s*18~1gand APP”-like immunoreactivities have been demonstrated in certain telencephalic areas such as the cortex, amygdala and the caudate nucleus. In the present study, we provide immunohistochemical evidence that these two peptides coexist in certain neurons in the rat telencephalon. EXPERIMENTAL Male Sprague-Dawley
albino
PROCEDURES rats (15&200
g) were used.
Abbreviations: APP, avian pancreatic polypeptide; cholecystokinin; FITC, fluoresceinisothiocyanate; phosphate buffered saline.
CCK, PBS,
439
Both untreated animals and rats, which had received an intraventricular injection of colchicine (120 pg/20 ~1 saline) 24 h prior to death, were examined. Animals were perfused through the ascending aorta with ice-cold 10% formalin. The brain and pancreas were dissected out and placed in the same fixative for 90min. After rinsing overnight in 0.1 M phosphate buffer (pH 7.4) containing 5% sucrose the tissues were cut on a cryostat (Dittes, Heidelberg, F.R.G.) at -20°C (section thickness 10 or 14 pm). The sections were processed for the indirect immunofluorescence procedure of Coons’ as previously described.’ Briefly, the sections were incubated at 4°C in a humid atmosphere for 24 h with antibodies raised in rabbits to either APP (J. M. Lundberg, K. Tatemoto, L. Terenius, T. Hokfelt & V. Mutt, in preparation) (diluted 1:200) or somatostatin’ (diluted 1: 100). They were then rinsed in phosphate buffered saline (PBS) and incubated in a 1: 10 dilution of fluoresceinisothiocyanate (FITC)-conjugated swine anti-rabbit IgG (Dako, Copenhagen, Denmark) for 30min at 37°C rinsed in PBS, mounted in glycerol-PBS (3: 1) and examined in a Zeiss fluorescence microscope. In certain experiments serial 10pm thick sections were stained alternately for either APP or somatostatin. In additional experiments, sections stained for APP were examined and photographed and then eluted with acid potassium permanganate3s and subsequently stained for somatostatin. When the anti-somatostatin serum was omitted as a control in the restaining experiments, no staining was observed after the second incubation with the FITClabelled serum, indicating that the APP antiserum had been successfully eluted. APP or somatostatin antiserum preabsorbed with an excess of APP and somatostatin (5Opg peptide/ml antiserum diluted l:lO), respectively, served as controls. In both cases, the specific staining described in this study was abolished. In contrast, preabsorption of the APP antiserum
440
S. R. Vincent
or trl.
sor.L4.‘5.3” Furthermore, a somatostatinand a gastrin/cholecystokinin (CCK)-like peptide coexist in the had no effect on the staining obtained. gastrointestinal tract.“’ an enkephalin- and an APPlike peptide coexist in the spinal cord,13 a substance P- and a thyrotropin-releasing hormone-like peptide RESULTS coexist, together with 5-hydroxytryptamine in medulIn agreement with the previous report of Loren ~‘r lary neurons” and a substance P- and a CCK-like u/..‘~ APP-positive neurons were observed in many peptide coexist in the periaquaductal mesencephalic areas of the telencephalon including the striatum. central gray.32 In none of the four latter cases has it neocortex, nucleus accumbens and olfactory tuberbeen demonstrated that the two peptides arc derived cule. Somatostatin-positive cell bodies were also obfrom a common precursor. It is interesting to note served in all these areas. Both of these peptides often that these four and the present example of coexistence had an intracellular distribution resembling that of are confined only to a population of neurons. For the Golgi apparatus, a point previously noted for example, some APP immunoreactive neurons do not somatostatin cells both in the brain and in peripheral appear to contain somatostatin-like immunoreactivity neurons.14 Sequential staining for APP followed by and ric,r ~w.w. and most substance P neurons do not elution and restaining for somatostatin. indicated that contain CCK-like immunorcactivity and r-ic,c,w~,w,~~ in the striatum these peptides were often contained in indicating that the coexistence in these instances canthe same neurons (Figs la-d). This could also be seen not be explained simply by a common precursor. It when serial sections were stained alternately for the may be speculated that neurons have the potential of two peptides (Figs le, f). Cells which were stained producing several peptides but that usually one. or as shown here. sometimes two peptides are expressed at positively for somatostatin in one section were often positively stained for APP in the adjacent section. the genetic lebel or derived from post-translational Using this procedure many cells in the neocortex processing. The functional role of peptides in the nervous sys(Figs lg, h), hippocampus (Figs 2a d), lateral septum. tem is still not fully understood. but a transmitter role nucleus accumbens and olfactory tubercle were also has been suggested.2’ Somatostatin has been reported found to contain the two peptides. to depress neuronal activity in the brain,2H possibly These peptides do not appear to coexist in all areas. rin an action on Ca2 ’ transport.33 The possibility however. This is illustrated in the case of the hypoexists that the APP-like peptide may also have a thalamus (Figs 2e, f), where numerous somatostatinneurotransmitter or neuromodulatory action in the positive cells are found in the anterior periventricular nervous system. The occurrence of more than one area. In the adjacent section. APP is seen only in peptide with modulatory function in a neuron might fibres with no positive cell bodies being present. Also, allow neurons to transmit more differentiated messin the pancreas APP and somatostatin were never ages than could be obtained by a single substance. found to coexist either in serial sections or in restainSimilar speculations have been advanced to explain ing experiments (Figs 3a-d). This is in agreement with the significance of another type of coexistence, i.e. earlier studies, which have indicated that the two pepcoexistence of a ‘classical’ transmitter. such as nortides are contained in different cell types of the panadrenaline, with a peptide. such as somatostatin. as creas.‘,*” first observed in peripheral sympathetic neuron? and in many other instances.‘“.” An additional peptide DISCUSSION may also serve to subdivide a transmitter-homogenic neuron population into subclasses. The results of this study indicate that certain APP has not as yet been isolated from mammalian neurons in the rat forebrain contain both somatostabrain and thus the exact chemical nature of the APPtin- and APP-like immunoreactivity. The preabsorpimmunoreactive substance observed in the present tion experiments demonstrate that each antiserum study is unknown. Loren PI tr/.2’ have pointed out does not cross-react with the other peptide. Also, the that neurons stained with antisera to APP are not observation that certain areas such as the perivenstained with antisera raised to either human or bovine tricular hypothalamus and the pancreas contain numerous strongly immunoreactive somatostatinpancreatic polypeptide. T~LIS. the APP-like immunopositive cells which are not stained by the APP antireactive substance in the mammalian nervous system is distinct from the endocrine pancreatic polypeptide serum. indicates that the two sera are not merely although both are recognized by our APP antiserum. recognizing a precursor common to both peptides. The present findings represent a further situation in A peptide somewhat similar to APP in structure has which two (or more) peptides coexist in the same been recently isolated from porcine intestine and neuron. Thus, it was demonstrated several years ago brain.34 but this peptide (PYY) does not appear to that some hypothalamic neurons contain both cross-react with the APP antiserum used in the adrenocorticotropic hormone P-endorphin and prcscnt study (J. M. Lundberg 01 rrl.. unpublished). (ACTH),3.2h.3h and it has been demonstrated that In conclusion, the present findings of peptide coexthese two polypeptides have a common precuristence in the mammalian telencephalon. together with
excess synthetic
sorption
somatostatin
of the somatostatin
or. alternatively,
antiserum
with
preab-
excess APP
Fig. la-h. lmmunofluorescence micrographs of the striatum (a-f) and neocortex (g, h) of a colchicine injected rat after incubation with antisera to avian pancreatic polypeptide (a, c, e, g) or somatostatin (b, d, f, h). a, b and c, d show two sections of the striatum stained for APP (a, c) and then eluted according to Tramu et al. 35 and restained for somatostatin (b, d). Arrows indicate APP-positive cells (a) not stained for somatostatin (b). e and f are serial 10/tm thick sections showing the same striatal neuron and g and h are serial 10 #m thick sections of the same cortical neuron (layer IV) stained for A P P (e, g) and somatostatin (f, h}. Arrow indicates a somatostatin-positive cell (h) not found in the adjacent section stained for A P P (g). Bar indicates 50/~m for all Figures. 441
Fig. 2~f. Immunofluorescence micrographs of the hippocampus (a-d) and the anterior periventricular hypothalamus (e, f) of a colchicine injected rat after incubation with antisera to avian pancreatic polypeptide (a, c, e) or somatostatin (b, d, f). a, b and c, d show two sections of the CA1 region of the dorsal hippocampus stained for APP (a, c) and then eluted according to Tramu et al. 35 and restained for somatostatin (b, d). p indicates the pyramidal cell layer (a) and the asterisk indicates a blood vessel (c, d). e and f are serial 10 #m thick sections showing APP-positive fibres (e) and somatostatin-positive cell bodies (f) adjacent to the third ventricle (asterisk). Bars indicate 50 #m for all figures. 442
Fig. 3 ~ d . Immunofluorescence micrographs of rat pancreas after incubation with antisera to avian pancreatic polypeptide (a, c), elution according to Tramu e t al. 35 followed by incubation with antisera to somatostatin (b, d). c and d are higher magnifications of the islet as indicated in a. The arrows indicate two cells, one of which contains A P P only (a, c) while the other cell is positive only for somatostatin (b, d). Note that the two cells have a similar shape but minor differences (arrow heads) can be seen. Superposition examination revealed that these two cells in fact are not identical but lie adjacent to each other. Asterisks indicate a blood vessel. Bars indicate 50 ~m for all Figures. 443
Coexistence
of somatostatin
with the previous examples of coexistence found in other areas, indicate that the occurrence of more than one
neuroactive
substance
common phenomenon nervous system.
within
throughout
Acknowledgements-The present study grants from the Swedish Medical
a single
cell
is a
many parts of the
was supported by Research Council
and APP
445
(04X-2887; 04X-3766), Knut och Alice Wallenbergs Stiftelse, Magnus Bergvalls Stiftelse, Sven och Ebba-Christina Hagbergs Stiftelse, Ollie och Elof Ericssons Stiftelse and the Swedish Tobacco Company. S.R.V. is a fellow of the Canadian Medical Research Council, L.S. is a fellow of the Swedish Medical Research Council. We thank MS B. Frideen and MS A. Lundmark for expert secretarial help.
REFERENCES G., Rothman J., Chang R., Fernando-Durango R., Elde R., Coy D. H., Meyers M. & Schally 1. Arimura A., Lundquist A. V. (1978) Radioimmunoassay of somatostatin. Metab. Clin. Exp. 24, Suppl.1, 1139-l 149. 2. Bennet-Clarke C., Romagnano M. A. &Joseph S. A. (1980) Distribution of somatostatin in the rat brain: telencephaIon and diencephalon. Brain Res. 188, 473-486. evidence that the same neurons in the 3. Bloch B., Bugnon C., Fellman D. & Lenys D. (1978) Immunocytochemical human infundibular nucleus are stained with anti-endorphins and antisera of other related peptides. Neuroscience Letters 10, 147-152. 4. Brazeau P., Vale W., Burgus R., Ling N., Butcher M., Rivier R. & Guillemin R. (1973) Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone. Science N.Y. 179, 77-79. 5. Coons A. (1958) Fluorescent antibody methods. In Genera/ C~~tochemical Methods (ed. Danielli J. F.) pp. 399-422. Academic Press, New York. 6. HGkfelt T., Efendii- S., Johansson 0.. Luft R. & Arimura A. (1974) Immunohistochemical localization of somatostatin (growth hormone release-inhibiting factor) in the guinea-pig brain. Brain Rrs. 80, 1655196. 7. Hiikfelt T., Elde R., Fuxe K., Johansson O., Ljungdahl ri., Goldstein M., Luft R., Efendii: S., Nilsson G., Terenius L.. Sauten D., Jeffcoate S. L., Rehfeld J.. Said S., Perez de la Mora M., Possan L., Tapia R., Tesan L. & Palacios R. (1978) Aminergic and peptidergic pathways in the nervous system with special reference to the hypothalamus. In Hypothalamus (eds. Reichlin S., Baldesarini R. J. & Martin J. B.) pp. 69-135. Raven Press, New York. 8. HBkfelt T., Elfvin L. G.. Elde R., Schultzberg M., Goldstein M. & Luft R. (1977) Occurrence of somatostatin-like immunoreactivity in some peripheral sympathetic noradrenergic neurons. Proc. mtn. Acad. Sci. U.S.A. 74, 3587-3591. 9. Hlikfelt T., Fuxe K., Goldstein M. & Joh T. H. (1973) Immunohistochemical localization of three catecholamine synthesizing enzymes: aspects on methodology. Histochemie 33, 231-254. 10. Hiikfelt T., Johansson O., Ljungdahl A., Lundberg J. M. & Schultzberg M. (1980) Peptidergic neurones. Nature. Lond. 284, 515-521. M.. Johansson O., Ljungdahl A & Rehfeld J. (1980) Coexistence of Peptides 11. HGkfelt T., Lundberg J. M., Schultzberg and Putative Transmitters in Neurons. In Neural Peprides and Neuronal Communication (eds Costa E. & Trabucchi M.) pp. l-23. Raven Press, New York. 12. Hiikfelt T., Lundberg J. M., Terenius L., Jancsb G. & Kimmel J. (1981) Avian pancreatic polypeptide (APP) immunoreactive neurons in the spinal cord and spinal trigeminal nucleus. Peptides 2, 81-87. M. & Kimmel J. (1981) Presence of avian pancreatic polypeptide-like 13. Hunt S. P., Emson P. C., Gilbert R.. Goldstein immunoreactivity in catecholamine and methionine-enkephalin-containing neurones within the central nervous system. Neuroscience Letters 21. 125-130. 0. (1978) Localization of somatostatin-like immunoreactivity in the Golgi apparatus of central and 14. Johansson peripheral neurons. Histochemie 58, 167-176. 15. Johansson 0. & Hakfelt T. (1980) Thyrotropin releasing hormone, somatostatin and enkephalin: distribution studies using immunohistochemical techniques. J. Hisrochem. Cyrochem. 28, 364366. 16. lohansson 0.. HGkfelt T.. Pernow B., Jeffcoate S. L., White N., Steinbusch H. W. M., Verhofstad A. A. J., Emson P. C. & Spindel E. (1981) Immunohistochemical support for three putative transmitters in one neuron: coexistence of 5_hydroxytryptamine, substance P- and thyrotropin releasing hormone-like immunoreactivity in medullary neurons projecting to the spinal cord. Neuroscience 6, 1857-1881. of a new pancreatic polypeptide 17. Kimmel J. R., Hayden L. J. & Pollock H. G. (1975) Isolation and characterization hormone. J. hiol. Chem. 250, 9369-9376. and extrahypothalamic distribution of somatostatin-immunoreactive elements in the 18. Kirsch B. (1978) Hypothalamic rat brain. Cell Tissue Rex 195, 499-513. H. (1980) An intermittant somatostatin-immunoreactivity in the cortex and basal ganglia of 19. Kirsch B. 8~ Leonhardt the rat. Cell Tissue Rex 205, 327-331. R., Pollock H. G. & Kimmel J. (1974) Localization of APP, a postulated new 20. Larsson L.-I., Sundler F., Hgkanson hormone, to a pancreatic endocrine cell type. Histochenlie 42, 377-382. R. & Sundler F. (1979) Immunoreactive pancreatic polypeptide (PP) occurs in the 21. Lorkn I., Alumets J., Hgkanson central and peripheral nervous system: preliminary immunocytochemical observations. Cell Tissue Res. 200, 179-186. J. M., HGkfelt T., .&ngg&rd A., Kimmel J., Goldstein M. & Markey K. (1980) Coexistence of an avian 22. Lundberg pancreatic polypeptide (APP) immunoreactive substance and catecholamine in some peripheral and central neurons. Acra physiol. stand. 110, 107-109. J. M., Hiikfelt T., Schultzberg M., Uvnis-Wallensten K., Kiihler C. & Said S. I. (1979) Occurrence of 23. Lundberg vasoactive intestinal polypeptide (VIP)-like immunoreactivity in certain cholinergic neurons of the cat: evidence from combined immunohistochemistry and acetylcholinesterase staining. Neuroscience 4. 1539-1559.
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24. Mams R. E., Eipper B. A. & Ling N. (1977) Common precursor to corticotropins and endorphins. Proc. natn. Acad. Sci. U.S.A. 74, 30143018. 25. Nakanishi S., lnoue A., Kita T., Nakamura M., Chang A. C. Y., Cohen S. N. & Numa S. (1979) Nucleotide sequence of cloned cDNA for bovine corticotropin-P-lipotropin precursor. Nature, Lond. 278, 423427. 26. Nilaver G.. Zimmerman E. A., Defendin R., Liotta A. S., Krieger D. T. & Brownstein M. J. (1979) Adrenocorticotropin and p-lipotropin in the hypothalamus; localization in the same arcuate neurons by sequential immunocytochemical procedures. J. Cell Biol. 81, 50-58. 27. Otsuka M. & Takahishi T. (1977) Putative peptide neurotransmitters. A. Reo. Phurmac. Toxicol. 17, 4255439. 2X. Renaud L. P.. Martin J. B. & Brazeau P. (1975) Depressant action of TRH, LH RH and somatostatin on activity of central neurons. Nature. Lond. 255, 233-235. 29. Richoux J. P. & Dubois M. P. (1980) Neuronal systems immunologically related to the somatostatin system in the garden dormouse. Cc// Tissue Rrs. 209, 4555472. 30. Roberts J. L. & Herbert E. (1977) Characterization of a common precursor to corticotropin and /&lipotropin peptides and their arrangement relative to corticotropin in the precursor synthesized in a cell-free system. Proc. natn. Acad. Sci. C’.S.A. 74, 5300-5304. 31. Schultzberg M.. Hokfelt T., Nilsson G., Terenius L., Rehfeld J. H., Brown M., Elde R., Goldstein M. 8~ Said S. (1980) Distribution of peptide and catecholamine-containing neurons in the gastrointestinal tract of rat and guinea-pig: immunohistochemical studies with antisera to substance P, vasoactive intestinal polypeptide, enkephalins, somatostatin. gastrinjcholecystokinin, neurotensin and dopamine fi-hydroxylase. Neuroscience 5, 687-744. 32. Skirboll L.. Hokfelt T., Rehfeld J. & Cuello A. C. (1981) Cholecystokinin/substance P immunoreactive neurons in the periaquaductal central grey project to the spinal cord. Neuroscience Letters, in press. 33. Tan A. T.. Tsang D., Renaud L. P. & Martin J. B. (1977) Effect of somatostatin on calcium transport in guinea-pig cortex synaptosomea Brain Res. 121, 193-196. 34. Tatemoto K. & Mutt V. (1980) Isolation of two novel candidate hormones using a chemical method for finding naturally occurring polypeptides. Nature, Land. 285, 417-418. 35. Tramu G.. Pillez A. & Leonardell J. (1978) An effective method of antibody elution for the successive or simultaneous localization of two antigens by immunocytochemistry. J. Histochem. C’ytochem. 26, 322-324. 36. Watson S.. Akil H.. Richard C. W. III & Barchas J. D. (1978) Evidence for two separate opiate peptide neuronal systems. Nuturr. Lond. 275, 226- 228. (Accepted
18 September
1981)
0306.4522:82/020439-07$03,00/O Pergamon Press Ltd 0 1982 IBRO
COEXISTENCE OF SOMATOSTATINAND AVIAN PANCREATIC POLYPEPTIDE (APP)-LIKE IMMUNOREACTIVITY IN SOME FOREBRAIN NEURONS S. R.
VINCENT*, L. SKIRBOLL*, T. H~KFELT*, 0. JOHANSSON*,J. M. LUNDBERG-~-, R. P. ELDE$, L. TERENIUS#and J. KIMMELT *Departments of Histology and tPharmacology, Karolinska Institutet, Stockholm, Sweden; IDepartment of Anatomy, University of Minnesota, Minneapolis, Minnesota, U.S.A.; $Department IMcllwain
of Pharmacology,
Laboratory,
Uppsala
The University
University.
of Kansas
Medical
Uppsala. Center,
Sweden; Kansas,
U.S.A.
Abstract-The indirect immunofluorescence technique was used to demonstrate the coexistence of somatostatin together with avian pancreatic polypeptide-like immunoreactivity within certain neurons of the rat forebrain. Numerous neurons containing these peptides were observed in the neocortex, hippocampus, olfactory tubercle, striatum, nucleus accumbens and lateral septum. In studies of serial sections stained alternately for these two peptides, and in restaining experiments, it could be determined that in many neurons in these areas these two peptides coexisted. In other brain areas such as the anterior periventricular hypothalamus, somatostatin cells were never found to contain avian pancreatic polypeptide-like immunoreactivity. Also, within the pancreas these two peptides were never found to coexist in the same cells. The findings represent a further example of the coexistence of more than one neuropeptide within a single neuron.
THE GROWTH hormone release-inhibiting hormone, somatostatin, was isolated and structurally characterized in 1973.4 Subsequent immunohistochemical studies demonstrated somatostatin-containing cells in the peripheral and central nervous systems including the gastro-intestinal tracts as well as in pancreas.6 Avian pancreatic polypeptide (APP) was originally isolated from chicken pancreas.” APP-like immunoreactivity in neurons was first demonstrated by Loren, Alumets, Hakanson & SundIe?’ and subsequently by several other groups.12~‘3*22 Both somatostatin and APP have been shown to coexist with either a classical transmitter or with another peptide in the same neuron. Thus, somatostatin-like immunoreactivity has been found in some peripheral sympathetic noradrenergic neurons.’ APP-like immunoreactivity has been shown to coexist with catecholamines in some central and peripheral neurons13.22 and with methionineenkephalin in sacral parasympathetic neurons.i3 Both somatostatin2~7~14~1s*18~1gand APP”-like immunoreactivities have been demonstrated in certain telencephalic areas such as the cortex, amygdala and the caudate nucleus. In the present study, we provide immunohistochemical evidence that these two peptides coexist in certain neurons in the rat telencephalon. EXPERIMENTAL Male Sprague-Dawley
albino
PROCEDURES rats (15&200
g) were used.
Abbreviations: APP, avian pancreatic polypeptide; cholecystokinin; FITC, fluoresceinisothiocyanate; phosphate buffered saline.
CCK, PBS,
439
Both untreated animals and rats, which had received an intraventricular injection of colchicine (120 pg/20 ~1 saline) 24 h prior to death, were examined. Animals were perfused through the ascending aorta with ice-cold 10% formalin. The brain and pancreas were dissected out and placed in the same fixative for 90min. After rinsing overnight in 0.1 M phosphate buffer (pH 7.4) containing 5% sucrose the tissues were cut on a cryostat (Dittes, Heidelberg, F.R.G.) at -20°C (section thickness 10 or 14 pm). The sections were processed for the indirect immunofluorescence procedure of Coons’ as previously described.’ Briefly, the sections were incubated at 4°C in a humid atmosphere for 24 h with antibodies raised in rabbits to either APP (J. M. Lundberg, K. Tatemoto, L. Terenius, T. Hokfelt & V. Mutt, in preparation) (diluted 1:200) or somatostatin’ (diluted 1: 100). They were then rinsed in phosphate buffered saline (PBS) and incubated in a 1: 10 dilution of fluoresceinisothiocyanate (FITC)-conjugated swine anti-rabbit IgG (Dako, Copenhagen, Denmark) for 30min at 37°C rinsed in PBS, mounted in glycerol-PBS (3: 1) and examined in a Zeiss fluorescence microscope. In certain experiments serial 10pm thick sections were stained alternately for either APP or somatostatin. In additional experiments, sections stained for APP were examined and photographed and then eluted with acid potassium permanganate3s and subsequently stained for somatostatin. When the anti-somatostatin serum was omitted as a control in the restaining experiments, no staining was observed after the second incubation with the FITClabelled serum, indicating that the APP antiserum had been successfully eluted. APP or somatostatin antiserum preabsorbed with an excess of APP and somatostatin (5Opg peptide/ml antiserum diluted l:lO), respectively, served as controls. In both cases, the specific staining described in this study was abolished. In contrast, preabsorption of the APP antiserum
440
S. R. Vincent
or trl.
sor.L4.‘5.3” Furthermore, a somatostatinand a gastrin/cholecystokinin (CCK)-like peptide coexist in the had no effect on the staining obtained. gastrointestinal tract.“’ an enkephalin- and an APPlike peptide coexist in the spinal cord,13 a substance P- and a thyrotropin-releasing hormone-like peptide RESULTS coexist, together with 5-hydroxytryptamine in medulIn agreement with the previous report of Loren ~‘r lary neurons” and a substance P- and a CCK-like u/..‘~ APP-positive neurons were observed in many peptide coexist in the periaquaductal mesencephalic areas of the telencephalon including the striatum. central gray.32 In none of the four latter cases has it neocortex, nucleus accumbens and olfactory tuberbeen demonstrated that the two peptides arc derived cule. Somatostatin-positive cell bodies were also obfrom a common precursor. It is interesting to note served in all these areas. Both of these peptides often that these four and the present example of coexistence had an intracellular distribution resembling that of are confined only to a population of neurons. For the Golgi apparatus, a point previously noted for example, some APP immunoreactive neurons do not somatostatin cells both in the brain and in peripheral appear to contain somatostatin-like immunoreactivity neurons.14 Sequential staining for APP followed by and ric,r ~w.w. and most substance P neurons do not elution and restaining for somatostatin. indicated that contain CCK-like immunorcactivity and r-ic,c,w~,w,~~ in the striatum these peptides were often contained in indicating that the coexistence in these instances canthe same neurons (Figs la-d). This could also be seen not be explained simply by a common precursor. It when serial sections were stained alternately for the may be speculated that neurons have the potential of two peptides (Figs le, f). Cells which were stained producing several peptides but that usually one. or as shown here. sometimes two peptides are expressed at positively for somatostatin in one section were often positively stained for APP in the adjacent section. the genetic lebel or derived from post-translational Using this procedure many cells in the neocortex processing. The functional role of peptides in the nervous sys(Figs lg, h), hippocampus (Figs 2a d), lateral septum. tem is still not fully understood. but a transmitter role nucleus accumbens and olfactory tubercle were also has been suggested.2’ Somatostatin has been reported found to contain the two peptides. to depress neuronal activity in the brain,2H possibly These peptides do not appear to coexist in all areas. rin an action on Ca2 ’ transport.33 The possibility however. This is illustrated in the case of the hypoexists that the APP-like peptide may also have a thalamus (Figs 2e, f), where numerous somatostatinneurotransmitter or neuromodulatory action in the positive cells are found in the anterior periventricular nervous system. The occurrence of more than one area. In the adjacent section. APP is seen only in peptide with modulatory function in a neuron might fibres with no positive cell bodies being present. Also, allow neurons to transmit more differentiated messin the pancreas APP and somatostatin were never ages than could be obtained by a single substance. found to coexist either in serial sections or in restainSimilar speculations have been advanced to explain ing experiments (Figs 3a-d). This is in agreement with the significance of another type of coexistence, i.e. earlier studies, which have indicated that the two pepcoexistence of a ‘classical’ transmitter. such as nortides are contained in different cell types of the panadrenaline, with a peptide. such as somatostatin. as creas.‘,*” first observed in peripheral sympathetic neuron? and in many other instances.‘“.” An additional peptide DISCUSSION may also serve to subdivide a transmitter-homogenic neuron population into subclasses. The results of this study indicate that certain APP has not as yet been isolated from mammalian neurons in the rat forebrain contain both somatostabrain and thus the exact chemical nature of the APPtin- and APP-like immunoreactivity. The preabsorpimmunoreactive substance observed in the present tion experiments demonstrate that each antiserum study is unknown. Loren PI tr/.2’ have pointed out does not cross-react with the other peptide. Also, the that neurons stained with antisera to APP are not observation that certain areas such as the perivenstained with antisera raised to either human or bovine tricular hypothalamus and the pancreas contain numerous strongly immunoreactive somatostatinpancreatic polypeptide. T~LIS. the APP-like immunopositive cells which are not stained by the APP antireactive substance in the mammalian nervous system is distinct from the endocrine pancreatic polypeptide serum. indicates that the two sera are not merely although both are recognized by our APP antiserum. recognizing a precursor common to both peptides. The present findings represent a further situation in A peptide somewhat similar to APP in structure has which two (or more) peptides coexist in the same been recently isolated from porcine intestine and neuron. Thus, it was demonstrated several years ago brain.34 but this peptide (PYY) does not appear to that some hypothalamic neurons contain both cross-react with the APP antiserum used in the adrenocorticotropic hormone P-endorphin and prcscnt study (J. M. Lundberg 01 rrl.. unpublished). (ACTH),3.2h.3h and it has been demonstrated that In conclusion, the present findings of peptide coexthese two polypeptides have a common precuristence in the mammalian telencephalon. together with
excess synthetic
sorption
somatostatin
of the somatostatin
or. alternatively,
antiserum
with
preab-
excess APP
Fig. la-h. lmmunofluorescence micrographs of the striatum (a-f) and neocortex (g, h) of a colchicine injected rat after incubation with antisera to avian pancreatic polypeptide (a, c, e, g) or somatostatin (b, d, f, h). a, b and c, d show two sections of the striatum stained for APP (a, c) and then eluted according to Tramu et al. 35 and restained for somatostatin (b, d). Arrows indicate APP-positive cells (a) not stained for somatostatin (b). e and f are serial 10/tm thick sections showing the same striatal neuron and g and h are serial 10 #m thick sections of the same cortical neuron (layer IV) stained for A P P (e, g) and somatostatin (f, h}. Arrow indicates a somatostatin-positive cell (h) not found in the adjacent section stained for A P P (g). Bar indicates 50/~m for all Figures. 441
Fig. 2~f. Immunofluorescence micrographs of the hippocampus (a-d) and the anterior periventricular hypothalamus (e, f) of a colchicine injected rat after incubation with antisera to avian pancreatic polypeptide (a, c, e) or somatostatin (b, d, f). a, b and c, d show two sections of the CA1 region of the dorsal hippocampus stained for APP (a, c) and then eluted according to Tramu et al. 35 and restained for somatostatin (b, d). p indicates the pyramidal cell layer (a) and the asterisk indicates a blood vessel (c, d). e and f are serial 10 #m thick sections showing APP-positive fibres (e) and somatostatin-positive cell bodies (f) adjacent to the third ventricle (asterisk). Bars indicate 50 #m for all figures. 442
Fig. 3 ~ d . Immunofluorescence micrographs of rat pancreas after incubation with antisera to avian pancreatic polypeptide (a, c), elution according to Tramu e t al. 35 followed by incubation with antisera to somatostatin (b, d). c and d are higher magnifications of the islet as indicated in a. The arrows indicate two cells, one of which contains A P P only (a, c) while the other cell is positive only for somatostatin (b, d). Note that the two cells have a similar shape but minor differences (arrow heads) can be seen. Superposition examination revealed that these two cells in fact are not identical but lie adjacent to each other. Asterisks indicate a blood vessel. Bars indicate 50 ~m for all Figures. 443
Coexistence
of somatostatin
with the previous examples of coexistence found in other areas, indicate that the occurrence of more than one
neuroactive
substance
common phenomenon nervous system.
within
throughout
Acknowledgements-The present study grants from the Swedish Medical
a single
cell
is a
many parts of the
was supported by Research Council
and APP
445
(04X-2887; 04X-3766), Knut och Alice Wallenbergs Stiftelse, Magnus Bergvalls Stiftelse, Sven och Ebba-Christina Hagbergs Stiftelse, Ollie och Elof Ericssons Stiftelse and the Swedish Tobacco Company. S.R.V. is a fellow of the Canadian Medical Research Council, L.S. is a fellow of the Swedish Medical Research Council. We thank MS B. Frideen and MS A. Lundmark for expert secretarial help.
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18 September
1981)