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Comparative Distribution of Pituitary Adenylate Cyclase-Activating Polypeptideand Vasoactive Intestinal Polypeptide Immunoreactivity in the Chicken Forebraina

KRISTEL PEETERS, LUC R. BERGHMAN, AND FRANS VANDESANDE

Laboratory for Neuroendocrinology and Immunological Biotechnology, Katholieke Universiteit Leuven, Zoological Institute, Naamsestraat 59, B-3000 Leuven, Belgium

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a relatively newmember of the secretin/glucagon/vasoactive intestinal polypeptide (VIP) family. Itwas identified and purified on the basis of its ability to stimulate the accumulationof cyclic adenosine monophosphate (cAMP) in rat anterior pituitary cell cultures.Originally, two forms of the peptide were isolated from the ovine hypothalamus, oneof 38 amino acids (PACAP-38) and a C-terminally truncated form of 27 residues(PACAP-27).1 The primary structure of PACAP is highly conserved during evolu-tion. PACAP immunoreactivity is found in high concentration in the central nervoussystem of mammals (sheep,2 rat,3 monkey, and human4) and amphibians (frog5) andin various other tissues such as testis and adrenal gland. However, in avian specieslittle is known about PACAP.

Using polyclonal rabbit and sheep antisera we investigated the distribution ofPACAP-containing neurons in the forebrain of juvenile female chickens intraven-tricularly injected with colchicine.

MATERIALS AND METHODS

Polyclonal antisera were generated in sheep and rabbits against two syntheticfragments, CysPACAP11-27 and CysPACAP24-38, conjugated to human α-globu-lin. These antisera were tested for specificity by antigen spot tests as well as by solidphase adsorption.

Four-week-old, ad libitum fed, female broiler chickens were anesthetized withnembutal and injected with colchicine into the lateral ventricle. After a survival timeof 2 days, the birds were perfused transcardially with heparinized physiological sa-line solution, followed by Bouin Hollande sublimate fixative solution. Paraffin sec-tions were made and alternate sections were processed for PACAP and VIPimmunocytochemical staining using a peroxidase-conjugated secondary antiserumor a biotinylated secondary antiserum followed by streptavidin-peroxidase. Diami-nobenzidine was used as the chromogen. Method specificity was assessed by substi-

aThis work was supported by grants from the Belgian Nationaal Fonds voor Wetenschap-pelijk Onderzoek (to K.P. and L.B.).

418 ANNALS NEW YORK ACADEMY OF SCIENCES

tution of the primary antiserum by nonimmune serum and sequential omission of thevarious steps in the staining procedure.

RESULTS AND CONCLUSION

PACAP- and VIP-immunostained perikarya and fibers were mainly observed inthe preoptic and supraoptic region and throughout the hypothalamus. These immu-no-cytochemical results are summarized in TABLE 1.

PACAP-immunoreactive (ir) cells are mainly located in the preoptic and su-praoptic region. However, many more VIP-containing fibers were observed in thisarea. In the hypothalamus, the largest group of VIP-ir neurons was found within thelateral hypothalamus, nucleus anterior medialis hypothalami, and nucleus ventrome-dialis hypothalami. A second group was observed in the nucleus infundibuli hypo-thalami, nucleus inferioris hypothalami, and nucleus mamillaris mediales. PACAPir cells were most abundant in the nucleus paraventricularis magnocellularis. Bothneuropeptides were found in the median eminence and posterior lobe of pituitary.

ACKNOWLEDGMENTS

The VIP antiserum was a gift from Dr. S. Bläkser.6

TABLE 1. A Comparison of the Distribution of Immunoreactive PACAP and VIP

Brain region PACAP VIP

Organum septi laterale (LSO) - + P

Nucleus septalis medialis (SM), nucleus septalis lateralis (SL) (+) F ++ F

Nucleus supraopticus, pars ventralis (SOv) ++ P + P, ++ F

Nucleus magnocellularis preopticus (MPO) ++ P, + F (+) P, ++ F

Nucleus suprachiasmaticus, pars medialis (SCNm) + P (+) P+ F

Nucleus anterior medialis hypothalami (AM) (+) P, F ++ P, F

Regio lateralis hypothalami (LHy) + P, (+) F ++ P, + F

Nucleus paraventricularis magnocellularis (PVN) ++ P (+) F (+) P++ F

Nucleus periventricularis hypothalami (PHN) + P, (+) F ++ F

Nucleus ventromedialis hypothalami (VMN) (+) F ++ P, F

Nucleus inferioris hypothalami (IH) (+) P, F ++ P, + F

Nucleus infundibuli hypothalami (IN) (+) P, F ++ P, F

Nucleus mamillaris medialis (MM) - ++ P

Eminentia mediana (ME) + F ++ F

Substantia grisea centralis (GCt) - ++ P

Symbols: - = no immunoreactivity; (+) = moderate; + = strong; ++ = very strong immunore-activity; F = fibers; P = perikaya.

419PEETERS et al.: PACAP AND VIP IMMUNOREACTIVITY

REFERENCES

1. ARIMURA A. 1992. Pituitary adenylate cyclase-activating polypeptide (PACAP):Discovery and current status of research. Regul. Pept. 37: 287–303.

2. KÖVES K., ARIMURA A., SOMOGYVARI-VIGH A., VIGH S. & MILLER J. 1990. Immu-nohistochemical demonstration of a novel hypothalamic peptide, pituitary adeny-late cyclase-activating polypeptide, in the ovine hypothalamus. Endocrinology127: 264–271.

3. KÖVES K., ARIMURA A., GÖRCS T.G. & SOMOGYVARI-VIGH A. 1991. Comparativedistribution of immunoreactive pituitary adenylate cyclase-activating polypeptideand vasoactive intestinal polypeptide in rat forebrain. Neuroendocrinology 54:159-169.

4. VIGH S., ARIMURA A., KÖVES K., SOMOGYVARI-VIGH A., SITTON J. & FERMIN C.D.1991. Immunohistochemical localization of the neuropeptide, pituitary adenylatecyclase-activating polypeptide (PACAP), in human and primate hypothalamus.Peptides 12: 313–318.

5. YON L., FEUILLOLEY M., CHARTREL N., ARIMURA A., CONLON J.M., FOURNIER A.& VAUDRY H. 1992. Immunohistochemical distribution and biological activity ofpituitary adenylate cyclase-activating polypeptide (PACAP) in the central nervoussystem of the frog Rana ridibunda. J. Comp. Neurol. 324: 485–499.

6. KUENZEL W.J. & BLÄHSER S. 1994. Vasoactive intestinal polypeptide (VIP)-con-taining neurons: Distribution throughout the brain of the chick with focus upon thelateral septal organ. Cell Tissue Res. 275: 91–107.