Autoradiographic distribution of pituitary adenylate cyclase activating polypeptide (PACAP) binding sites in the rat brain

Neuroscience Letters, 126 (1991) 1033106 0 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$03.50

103

ADONIS 030439409 100237Y

NSL 07725

Autoradiographic distribution of pituitary adenylate cyclase activating polypeptide (PACAP) binding sites in the rat brain Yoshinori

Masuol,

Tetsuya

Ohtakil,

Yasushi Masudai, Yasuo Nagai2, Masahiro and Masahiko Fujino’

Sung,

Masao Tsuda’

I Tsukuba Research Laboratories, Takeda Chemical Industries, Ltd., Ibaraki (Japan) and 2Bioiogy Research Laboratories, Takeda Chemical Industries, Ltd., Osaka (Japan)

(Received 26 December 1990; Revised version received 28 January 1991; Accepted 30 January 1991) Key words;

Pituitary adenylate cyclase activating polypeptide (PACAP); Autoradiography;

Rat brain; Hippocampus; Pituitary; Mesencephalon

Distribution of pituitary adenylate cyclase activating polypeptide (PACAP) binding sites was investigated in the rat brain and pituitary gland by means of in vitro autoradiography. High densities of specific [rZ51]PACAPbinding were observed in the anterior pituitary, hippocampus (CA14 and dentate gyrus) and in the superior colliculus. Moderate to high labeling was observed in the periaqueductal gray matter, substantia nigra pars compacta, and in the habenula. The hypothalamus, thalamus, ventral tegmental area (VTA), mammillary body and medial geniculate body were moderately labeled. The present results support possible actions of PACAP on the pituitary functions, and further suggest that PACAP is a neurotransmitter/neuromodulator in the central nervous system.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a 38 amino acid peptide originally isolated from ovine hypothalamus [12]. Its N-terminal (l-28) sequence shows a 68 % homology with vasoactive intestinal polypeptide (VIP) [ 121. Subsequently, the 27-amino acid form of PACAP (PACAP27) was isolated from ovine hypothalamus, and found to have potent biological activity comparable to PACAP [13]. These two forms are suggested to be derived from a single 176-amino acid precursor [7]. Although the physiological role of PACAP is not clearly known, it was demonstrated that PACAP was 1000 times more potent than VIP in stimulating adenylate cyclase in rat pituitary cells [ 121and in PC 12h cells

P81. Immunohistochemical studies revealed the presence of PACAP-like immunoreactivity in the hypothalamus of the sheep [LX], monkey and human [16], and in the sheep posterior pituitary [8]. Specific PACAP binding sites which scarcely interact with VIP have been found in astrocytes [ 151, pancreatic acinar cells [3] and in the rat anterior pituitary [5]. Moreover, Ohtaki et al. [14] identified a PACAP receptor molecule in bovine brain fraction and demonstrated that it was obviously distinct from a

Correspondence: Y. Masuo, Tsukuba Research Laboratories, Takeda Chemical Industries, Ltd., 7 Wadai, Tsukuba, Ibaraki, 300-42 Japan,

brain VIP receptor. However, the topographical distribution of PACAP binding sites has not yet been demonstrated in the central nervous system (CNS). The aim of the present study was to investigate distribution of PACAP binding sites in the rat CNS by means of in vitro autoradiography. Five male Sprague-Dawley rats (3OOg) were used in this study. Animals were sacrificed by decapitation, and their brains immediately removed. Each pituitary gland was also removed and put on the brain in the original position. The brains with pituitaries were frozen with dry ice. This material was cut into 20-p-thick sections on a cryostat at - 15°C mounted onto gelatin-coated slide glasses, and stored at -80°C until experiment. Tissue sections were processed to [‘251]PACAP binding as described previously [14]. Briefly, sections were incubated for 60 min at 22°C with 0.1 nM [‘251]PACAP27 (2000 Ci/ mmol) [14] in phosphate-buffered saline (PBS), pH 7.4, containing 1 mM MgClz, 0.5 mM CaCl2,O. 1% bovine serum albumin, 0.1 mM PMSF, 0.1 pg/ml pepstatin and 0.05% CHAPS. Non-specific binding was determined in the presence of 1 ,uM unlabeled PACAP27. After incubation, sections were washed 3 times for 2 min in PBS at 4”C, and dipped in cold distilled water. In some experiments, tissue sections were wiped off from the slides with glass fiber filters (Whatman GF/F), and the radioactivity was measured in a gamma counter. For autoradiography, sections were dried with a cold stream of air. Film

c C A2

105

autoradiograms were obtained by the apposition of radiolabeled sections to Hyperfllm-3H (Amersham, Japan) at room temperature in the dark. Following 7 days of exposure, films were developed with Kodak D-19, rinsed in water and fixed with Kodak fixer. The radioactivity content of whole brain sections at the level of the diencephalon indicated that 75 s of [1251]PACAP binding was specific under our experimental conditions. In the kinetics of specific [‘251]PACAP27 association to slide-mounted tissue sections, specific binding increased time-dependently and reached a steady state after 60 min at 22°C. Moreover, 10e6 M VIP did not displace [1251]PACAP binding (data not shown). As shown in Fig. IA, the hippocampus was highly labeled with [‘251]PACAP. The binding sites were dense in thin layers of CA1-4 and dentate gyrus. Moderate to high densities of [1251]PACAPwere observed in the habenula. The hypothalamus and thalamus were moderately labeled. At the level of the mesencephalon (Fig. IB), [1251]PACAP binding sites were highly located in the anterior pituitary and superior colliculus in addition to the hippocampus. Moderate to high labeling was found in the periaqueductal gray matter and substantia nigra pars compacta. The ventral tegmental area (VTA), mammillary body and medial geniculate body were moderately labeled. In the present study, we demonstrate for the first time the autoradiographic distribution of PACAP binding sites in the rat brain and pituitary. The present study extends previous binding studies using tissue membranes [5, 141, since autoradiography of PACAP binding sites revealed topographic localization of PACAP binding sites in several brain regions which were not investigated. The most striking localization of PACAP binding sites was observed in the anterior lobe of the pituitary gland. This finding is in agreement with previous studies [12] showing that PACAP stimulates the accumulation of intracellular and extracellular CAMP in monolayer cultures of rat anterior pituitary cells. Thus, physiological PACAP receptors may be highly located in the anterior pituitary. In the brain, high densities of PACAP binding sites were observed in the hippocampus which receives cholinergic afferents from the septum and diagonal band [ 171.

Interestingly, it was previously demonstrated that VIP could modify cholinergic activity [9] in this structure. Since PACAP is structurally related to VIP, PACAP/ acetylcholine interaction should be investigated in the hippocampus. On the other hand, moderate to high densities of PACAP binding sites were observed in the substantia nigra pars compacta and VTA which contain a great population of dopaminergic neurons [2]. It was reported that VIP could modify dopamine metabolism in the mesencephalon [6]. Therefore, PACAP/dopamine interactions should be also investigated in the CNS. In this context, it would be of interest to study subregional distribution of PACAP binding sites in the mesencephalit regions. Lesion study may be available to investigate such neuropeptide/classical transmitter interactions [ 10, 1I]. Moreover, the present results reveal that a detailed topographical distribution of PACAP binding sites is somewhat different from that of VIP demonstrated previously [I]. Indeed, in the hippocampal formation, PACAP binding sites were highly located in thin layers of both CA14 and dentate gyrus, while VIP binding sites were concentrated in the dentate gyrus only. The substantia nigra pars compacta was highly labeled with PACAP but not with VIP, and binding sites for VIP but not for PACAP were dense in the thalamus. Localizations of binding sites for PACAP and VIP should be compared under identical experimental conditions. The present study demonstrated heterogeneous distribution of PACAP binding sites in the rat CNS, suggesting that PACAP is a neurotransmitter/neuromodulator in the CNS. These data may prompt further investigations on the function of PACAP in several brain regions. For instance, since it has been demonstrated that septohippocampal cholinergic neurons play an important role in memory [4], it would be of interest to study the physiological role for PACAP in mechanisms of memory. 1 Besson,

J., Sarrieau,

Rostene,

A., Vial, M., Marie,

W., Characterization

of vasoactive

intestinal

J.C.,

Rosselin,

and autoradiographic

peptide

binding

G. and

distribution

sites in the rat central

ner-

vous system, Brain Res., 398 (1986) 329336. 2 Bjiirklund,

A. and Lindval,

the CNS.

In A. Bjiirklund

Chemical

Neuroanatomy,

O., Dopamine-containing and T. Hokfelt Vol. 2, Classical

CNS, Part 1, Elsevier, Amsterdam,

(Eds.),

systems

in

Handbook

Transmitters

of

in the

1984, pp. 55-122.

Fig. 1. Autoradiogram were incubated

of [i25I]PACAP

binding

sites in rat frontal

section

at the level of the diencephalon

with 0.1 PM [iZ51]PACAP27 for 60 min at 22°C. Since sodocolour

ponds to the red areas, and the lowest density reflects the blue areas. Data obtained hippocampus ductal

CAl4;

gray matter;

DG, dentate PP, posterior

gyrus; Hab, Habenula; pituitary;

SC, superior striatum;

Hyp, hypothalamus; colliculus;

with 5 animals

MB, mammillary

SNc, substantia

Thal, thalamus;

(A) and mesencephalon

was used, the highest density of [i2?]PACAP27

VTA, ventral

were reproducible.

tegmental

AP, anterior

body; MG, medial geniculate

nigra pars compacta; area.

binding

SNr, substantia

(B). Sections sites corres-

pituitary;

CAl4,

body; PG, periaque-

nigra pars reticulata;

Str,

3 Buscail,

L., Gourlet,

mura. A.. Miyata,

P., Cauvin,

A., De Neef, P., Gossen,

A., Coy, D.H.,

Robberecht.

J., Presence of highly selective receptors nylate cyclase activating

peptide)

D., Ari-

P. and Christophe,

for PACAP

in membranes

(pituitary

ade-

from the rat pan-

creatic acinar cell line AR 4-25, FEBS Lett., 262 (1990) 77-81. 4 Gage, F.H., Bjorklund, tal grafts ameliorate

A. and Stenevi, V., Intrahippocampal

learning

sep-

in aged rats, Science, 225 (1984) 533-

lesions on binding

peptide in the rat nucleus accumbens

tegmental 12 Miyata,

sites for calcitonin

area, J. Chem. Neuroanat., A., Arimura,

and calciand ventral

in press.

A., Dahl, R.R.. Minamino,

N., Uehara,

A..

Jiang, L.. Culler, M.D. and Coy, D.H., Isolation

of a novel 38 resi-

due-hypothalamic

adenylate

in pituitary

polypeptide

cells, Biochem.

which stimulates Biophys.

Res. Commun.,

cyclase

164 (1989)

5677574.

536. i Gottschall.

P.E., Tatsuno,

terization

and distribution

peptide,

pituitary

crinology, c, Itoh.

Effects of cerebral tonin gene-related

I27

A. and Arimura,

of binding

adenylate

13 Miyata,

A., Charac-

cyclase-activating

polypeptide,

peptide

Endo-

G. and Takashima, on dopaminergic

Biochem.

A., Effect of vasoactive

system

in the rat brain,

Pep-

C., Ohkubo.

S., Ogi, K., Hosoya,

A., Jiang,

Fujino,

M.. A novel peptide

cDNAs,

cloning

which

Biophys.

Res. Commun., demonstration

adenylate

ovine hypothalamus.

cyclase:

15 Tatsuno,

166 (1990) 81-89.

polypeptide,

in the

Neurochem.,

by vasoactive

intestinal

peptide,

J.

42 (1984) 1131-l 134.

IO Masuo, Y., Pelaprat, t&e. W., Regulation striatum

Biophys.

D., Montagne, nigra.

S., Bouizar,

D. and Ros-

neurons

Effects of unilateral

with 6-hydroxydopamine on neurotensin content \;tes density, Brain Res., 510 (1990) 2033210.

11 MXUO. Y., Giscard-Dartevelle,

I.,

Gottschall,

in the rat

nigral

lesion

and its binding

Z. and Rostene,

P.E..

lase activating

polypeptide

Res. Commun.,

and Fermin,

CD.,

K&es,

poly-

and

Arimura.

A..

sites for pituitary

adenylate

cyc-

(PACAP)

K.

in rat astrocytes,

A., K&es,

K., Somogyvari-Vigh,

Immunohistochemical

(PACAP),

activating

171 (1990) 8388844.

Biochem.

168 (1990) 1027.-1033.

neuropeptide,

polypeptide

cyclase

M..

identifi-

pituitary

in human

A.. Sitton, J.

demonstration adenylate

and primate

cyclase

of a novel activating

hypothalamus,

Pep-

tides, in press. I., The hippocampus.

Neuroanatomy,

T., Ohtaki,

Adrenal

pheochromocytoma

nylate

cyclase

In P.C.

Raven. New York,

18 Watanabe,

Commun., W..

adenylate

C., Tsuda,

M., Molecuhir

Res. Commun.,

of specific binding

Biophys.

Y., Kitada.

A. and Fujino,

for pituitary Biophys.

adeny-

(PACAP38),

170 (1990) 6433648.

T.. Ishibashi,

Demonstration

17 Walaas, M.N., Scherman,

of neurotensin-containing

and substantia

Biochem.

K., Fujino,

of a neuropeptide

with 38 residues

Res. Commun..

P.E., Arimura.

C., Kubo,

27 residues of the pituitary

polypeptide

T., Watanabe, of receptor

Kitada,

A., Isolation

to the N-terminal activating

hypothalamic

I27 (1990) 264271.

L., Dahl, R.D.,

N. and Arimura,

16 Vigh, S., Arimura,

of a novel hypothalamic

0 Luine. V.N., Rostene, W., Rhodes, J. and McEwen, B.S., Activation of choline acetyltransferase

cation peptide,

A., Vigh, S. and Miller,

cyclase-activating

Endocrinology,

adenylate

H..

A. and

of the ovine and human

A., Somogyvari-Vigh,

J.. Immunohistochemical

Arimura.

stimulates

and characterization

Biochem.

pituitary

M., Itoh, Y., Onda,

L., Dahl. R.R., Stibbs, H.H.,

k Kiives. K.. Arimura, peptide.

14 Ohtaki,

Gottschall,

Miyata. molecular

corresponding late cyclase

tides,9(1988)315-317. 7 Kimura,

A., Jiang,

M., Minamino,

sites for the hypothalamic

(1990)272-277.

S.. Katsuura,

intestinal

I., Miyata,

T., Kitada,

activating

Emson

C.. Tsuda.

Chemical

M. and Fujino.

PCI 2h cells respond polypeptide.

173 (1990) 252-258.

(Ed.),

1983, pp. 337-358.

Biochem.

M.,

to pituitary

ade-

Biophys.

Res.