Distribution of pituitary adenylate cyclase-activating polypeptide and pituitary adenylate cyclase-activating polypeptide type I receptor mRNA in the chicken brain

Distribution of Pituitary AdenylateCyclase-Activating Polypeptide and

Pituitary Adenylate Cyclase-ActivatingPolypeptide Type I Receptor mRNA in

the Chicken Brain

KRISTEL PEETERS,* HELGA H.J. GERETS, LUTGARDE ARCKENS, AND

FRANS VANDESANDE

Laboratory of Neuroendocrinology and Immunological Biotechnology, Catholic Universityof Leuven, B-3000 Leuven, Belgium

ABSTRACTTo map in detail the brain areas in which pituitary adenylate cyclase-activating polypep-

tide (PACAP) may play a significant role in birds, the distribution of PACAP and PACAP typeI receptor (PAC1-R) mRNA was examined throughout the entire chicken brain by using insitu hybridization histochemistry. Widespread distribution of both PACAP and its receptormRNA was found. The telencephalic areas where the most intense signals for PACAP mRNAwere found included the hyperstriatum accessorium, the hippocampus, and the archistria-tum. In the diencephalon, a group of neurons that highly expressed PACAP mRNA wasobserved from the anterior medial hypothalamic nucleus to the inferior hypothalamic nu-cleus. Moderate expression was found in the paraventricular nucleus and the preoptic region.A second large group of neurons containing PACAP message was found within the nucleusdorsolateralis anterior thalami and extended caudally to the area around the nucleus ovoi-dalis and the nucleus paramedianus internus thalami. Furthermore, expression of PACAPmessage was observed within the bed nucleus of the pallial commissure, nucleus spiriformismedialis, optic tectum, cerebellar cortex, olfactory bulbs, and several nuclei within thebrainstem (dorsal vagal and parabrachial complex, reticular formation). The highest expres-sion of PAC1-R mRNA was found in the dorsal telencephalon, olfactory bulbs, lateral septum,optic tectum, cerebellum, and throughout the hypothalamus and thalamus. The presence ofPACAP and PAC1-R mRNA in a variety of brain areas in birds suggests that PACAPmediates several physiologically important processes in addition to regulating the activity ofthe pituitary gland. J. Comp. Neurol. 423:66–82, 2000. © 2000 Wiley-Liss, Inc.

Indexing terms: in situ hybridization; bird; central nervous system; expression; neuropeptide

Originally, pituitary adenylate cyclase-activatingpolypeptide (PACAP) was identified and purified fromovine hypothalami on the basis of its potent ability tostimulate the accumulation of cyclic adenosine monophos-phate (cAMP) in cultured rat pituitary cells. Two molecu-lar forms of the peptide have been isolated, a 38-aminoacid peptide (PACAP-38; Miyata et al., 1989) and aC-terminal truncated, 27-amino acid molecule (PACAP-27; Miyata et al., 1990).

The complementary DNAs (cDNAs) encoding for theprecursor of PACAP in human (Ohkubo et al., 1992),sheep (Kimura et al., 1990), and rat (Ogi et al., 1990) havebeen cloned, and analysis of the deduced amino acid se-

quences indicates that the structure of the peptide hasbeen fully conserved in mammals. Nonmammalian verte-brate cDNAs or genes encoding the PACAP precursorhave been cloned from catfish (McRory et al., 1995),

Grant sponsor: Queen Elisabeth Medical Foundation; Grant sponsor:ISERM.

*Correspondence to: Kristel Peeters, Laboratory of Neuroendocrinology andImmunological Biotechnology, Catholic University of Leuven, Naamsestraat59, B-3000 Leuven, Belgium. E-mail: [email protected].

Received 5 October 1999; Revised 14 March 2000; Accepted 14 March2000

THE JOURNAL OF COMPARATIVE NEUROLOGY 423:66–82 (2000)

© 2000 WILEY-LISS, INC.

Abbreviations

AA archistriatum anteriorAc nucleus accumbensAId archistriatum intermedium, pars dorsalisAIv archistriatum intermedium, pars ventralisAL ansa lenticularisAm archistriatum medialeAM nucleus anterior medialis hypothalamiAn nucleus angularisAp archistriatum posteriorAPH area parahippocampalisAVT area ventralis of TsaiBas nucleus basalisCA anterior commissureCb cerebellumCbI nucleus cerebellaris internusCbL nucleus cerebellaris lateralisCDL area corticoidea dorsolateralisCO chiasma opticumCPi cortex piriformisDLA nucleus dorsolateralis anterior thalamiDLAl nucleus dorsolateralis anterior thalami, pars lateralisDLAm nucleus dorsolateralis anterior thalami, pars medialisDLP nucleus dorsolateralis posterior thalamiDMP nucleus dorsomedialis posterior thalamiE ectostriatumEW nucleus of Edinger-WestphalFA tractus frontoarchistriaticusFDB fasciculus diagonalis BrocaeFL field LFLM fasciculus longitudinalis medialisFPL fasciculus prosencephali medialis (lateral forebrain bundle)FRL formatio reticularis lateralis mesencephaliFRM formatio reticularis medialis mesencephaliGCt substantia grisea centralis (central gray)GLdp nucleus geniculatis lateralis, pars dorsalis principalisGLv nucleus geniculatis lateralis, pars ventralisHA hyperstriatum accessoriumHD hyperstriatum dorsaleHIS hyperstriatum intercalatum supremumHL nucleus habenularis lateralisHM nucleus habenularis medialisHp, HP hippocampusHV hyperstriatum ventraleICo nucleus intercollicularisICT nucleus intercalatus thalamiIH nucleus inferioris hypothalamiImc nucleus isthmi, pars magnocellularisIN nucleus infundibuli hypothalamiINP nucleus intrapeduncularisIP nucleus interpeduncularisIpc nucleus isthmi, pars parvocellularisL lingulaLA nucleus lateralis anterior thalamiLhy lateral hypothalamic regionLLi nucleus lemnisci lateralis, pars intermediaLoC locus ceruleusLPO lobus parolfactoriusLSO lateral septal organME median eminenceML nucleus mamillaris lateralisMLd nucleus mesencephalicus lateralis, pars dorsalisMM nucleus mamillaris medialisMnVII nucleus motorius nervi facialisMnX nucleus motorius dorsalis nervi vagiMPO nucleus magnocellularis preopticusN neostriatumnBOR nucleus opticus basalisNC neostriatum caudalenCPa bed nucleus pallial commissureNI neostriatum intermediumnI nucleus intramedialisNIII nervus oculomotorius

nIX–X nucleus nervi glossopharyngei et nucleus motorius dorsalisnervi vagi

nTSM nucleus tractus septomesencephalicusnVI nervus abducensOI nucleus olivaris inferiorOM tractus occipitomesencephalicusOS nucleus olivaris superiorOV nucleus ovoidalisPA paleostriatum augmentatum (caudate putamen)PB nucleus parabrachialisPHN nucleus periventricularis hypothalamiPL nucleus pontis lateralisPM nucleus pontis medialisPMI nucleus paramedianus internus thalamiPMM nucleus premamillarisPOA preoptic areaPOM nucleus preopticus medialisPOP nucleus preopticus periventricularisPP paleostriatum primitivum (globus pallidus)PT nucleus pretectalisPTM nucleus pretectalis medialisPVN paraventricular nucleusPVO paraventricular organPVT paleostriatum ventraleQF tractus quintofrontalisR raphe nucleusRgc nucleus reticularis gigantocellularisRL nucleus reticularis lateralisROT nucleus rotundusRP nucleus reticularis pontis caudalisRPaM nucleus reticularis paramedianusRpc nucleus reticularis parvocellularisRPgc nucleus reticularis pontis caudalis, pars gigantocellularisRPgl nucleus reticularis paragigantocellularis lateralisRST nucleus reticularis subtrigeminalisRu nucleus ruberS nucleus solitariusSAC stratum album centraleSCd nucleus subceruleus dorsalisSCE stratum cellulare externumSCNm medial suprachiasmatic nucleusSCv nucleus subceruleus ventralisSGC stratum griseum centraleSGFS stratum griseum et fibrosum superficialeSGP stratum griseum periventriculareSL nucleus septalis lateralisSLu nucleus semilunarisSM nucleus septalis medialisSO stratum opticumSON supraoptic nucleusSP nucleus subpretectalisSpL nucleus spiriformis lateralisSpM nucleus spiriformis medialisSRt nucleus subrotundusT nucleus triangularisTDV nucleus et tractus descendens nervi trigeminiTn nucleus taeniaeTPc nucleus tegmenti pedunculopontinus, pars compacta (sub-

stantia nigra)TPO area temporoparietooccipitalisTrO tractus opticusTSM tractus septomesencephalicusVC ventriculus cerebelliVe nucleus vestibularisVeD nucleus vestibularis descendensVeL nucleus vestibularis lateralisVeM nucleus vestibularis medialisVIII third ventricleVL lateral ventricleVLT nucleus ventrolateralis thalamiVMN nucleus ventromedialis hypothalamiVT ventriculus tecti mesencephali

67PACAP AND PAC1-R mRNA IN CHICKEN BRAIN

salmon (Parker et al., 1993, 1997), and chicken (McRory etal., 1997) brain cDNA libraries. Moreover, a tunicatePACAP cDNA has been cloned (McRory and Sherwood,1997). The deduced amino acid sequences are highly ho-mologous to mammalian PACAP. In mammals, PACAPand a PACAP-related peptide (PRP) are encoded on onegene, whereas growth hormone-releasing hormone(GHRH) is on a separate gene. PACAP and PRP are re-lated structurally to GHRH, and all three peptides aremembers of a superfamily of peptides that includes gluca-gon, vasoactive intestinal polypeptide (VIP), peptide his-tidine methionine amide (PHM), glucose-dependent insu-linotropic polypeptide (GIP), and secretin. In contrast, infish (Parker et al., 1993, 1997; McRory et al., 1995),chicken (McRory et al., 1997), and invertebrates (McRoryand Sherwood, 1997), PACAP and a GHRH-like peptideappear to be encoded on the same gene.

The high degree of sequence conservation of PACAPsupports the concept that PACAP plays an importantphysiologic role. PACAP has been identified in neuronalelements of many brain areas as well as in fibers of severalorgans, including the gastrointestinal tract, the respira-tory tract, the adrenal glands, the pancreas, and the go-nads. This broad tissue distribution suggests pleiotropicactivities of the peptide. A multifunctional role of PACAPis suggested further by the presence of high-affinity bind-ing sites in these target tissues. PACAP produces its bio-logic effects by interacting with at least three types ofreceptors, type I PACAP receptors, which exhibit a highbinding affinity for PACAP-27 and PACAP-38 but not forVIP, and type I and II PACAP/VIP receptors, which do notdistinguish between PACAP and VIP (Arimura, 1998).Three receptor genes have been cloned in mammals, withPAC1-R corresponding to the type I binding sites (Hashi-moto et al., 1993, 1996b; Hosoya et al., 1993; Morrow etal., 1993; Ogi et al., 1993; Miyamoto et al., 1994) andVPAC1-R and VPAC2-R (Usdin et al., 1994) correspondingto type II sites (Harmar et al., 1998). At the pituitary level,under certain conditions, PACAP stimulates the release ofseveral pituitary hormones, including luteinizing hor-mone (LH), growth hormone (GH), adrenocorticotropin(ACTH), prolactin (PRL), and interleukin 6 (Il-6) fromfolliculostellate cells (Rawlings, 1994). Within the centralnervous system (CNS), PACAP not only is believed to havea role as a hypophysiotropic hormone, but it also has beensuggested that it acts as a neurotransmitter or neuro-modulator and a vasoregulator. Furthermore, PACAP wasshown to promote neurite outgrowth, to stimulate cellproliferation, and to prevent naturally programmed neu-ronal cell death. In addition, PACAP triggers the releaseof insulin and glucagon, activates steroidogenesis, andexerts a potent relaxant activity on gastrointestinal, bron-chial, and vascular smooth muscle fibers (Arimura, 1998).

Until now, almost nothing has been known about thepeptide within avian species. To determine whetherPACAP may play a significant role in the avian CNS, wemapped PACAP and PAC1-R gene expression in thechicken brain by using in situ hybridization (ISH).

MATERIALS AND METHODS

Animals and tissue preparation

Female broiler chickens (Hybros), 2.5 weeks of age, ob-tained from Euribrid (Aarschot, Belgium) were used in the

study. The animals were housed in large rooms and hadaccess to food and water ad libitum. Chicks were exposedto a constant photoperiod of 23 hours of light and 1 hour ofdark. For the ISH experiments, the birds were decapi-tated, and the brains were removed rapidly from the skulland frozen immediately by immersion in isopentanecooled on dry ice. Frozen brains were stored at 270°Cuntil they were sectioned. Twenty-micrometer frontal andsagittal sections were cut on a cryostat (Leitz, Van Hop-plynus, Belgium), thaw mounted onto 0.1% poly-L-lysine-coated slides (Sigma, Bornem, Belgium), and stored at220°C until they were processed for ISH. All experimen-tal procedures were in agreement with the Belgian lawson the Protection and Welfare of Animals and the Protec-tion of Experimental Animals and according to Interna-tional Guiding Principles for Biomedical Research Involv-ing Animals published by the Council for InternationalOrganizations of Medical Science.

ISH

Oligonucleotide probes. A 45-mer oligonucleotideprobe was used for the detection of PACAP mRNA. Thesequence was derived from bases 5,752–5,796 of thechicken GHRH/PACAP gene sequence (59-TTTCCGG-TAGCGGCTGTAGCTGTCCGTGAAGATGCCGTCTATG-TG-39; supplied by Eurogentec, Seraing, Belgium), whichencodes the first 15 amino acids of chicken PACAP(McRory et al., 1997). Two oligonucleotide probes wereused for the detection of PAC1-R mRNA: The sequence ofthe first probe was derived from bases 1,552–1,597(chPR1: 59-CGTTCACTCCGCTGCTCGCTAGAGAAGG-ATGACGATGTTTGAAATC-39), which is part of theC-terminal cytoplasmic tail of the receptor and is totallydifferent from the same region of the VPAC1-R, VPAC2-R,and other receptors of the VIP-secretin-PTH receptor sub-family. The second probe (chPR2: 59-GTTTATCATG-ATTGACCCAACCACAGGACCCTTGATCACCCACC-39)was derived from bases 1,106–1,123 of the chickenPAC1-R cDNA sequence (Peeters et al., 1999).

The oligodeoxynucleotide probes were 39-end labeled byusing terminal deoxynucleotidyl transferase (tdT; GibcoLife Technologies, Merelbeke, Belgium) and [35S]-dATP(NEN Life Science Products, Zaventem, Belgium). Free[35S] nucleotides were separated from the radioactiveprobes by using Nensorb™ 20-nucleic-acid purificationcartridges (NEN Life Science Products).

ISH histochemistry. Frozen, slide-mounted sectionswere thawed at room temperature and fixed subsequentlyfor 30 minutes in 4% paraformaldehyde in 0.1 Mphosphate-buffered saline, pH 7.4 (PBS), at 4°C. Sectionswere then rinsed twice for 10 minutes each in PBS at 4°Cand dehydrated through a series of ethanol: 60% for 1minute, 80% for 1 minute, 95% for 1 minute, and 100% for2 minutes. Sections were delipidated by immersion inchloroform for 5 minutes followed by 100% ethanol for 1minute and 95% ethanol for 1 minute. After air drying,sections were incubated overnight at 42°C in a humidchamber with approximately 106 cpm of [35S]-labeledprobe in 500 ml of a solution containing 50% formamide,43 standard saline citrate (SSC), 13 Denhardt’s reagent,1% N-lauryl-sarcosine, 10% dextran sulfate, 20 mMNaHPO4, pH 7.4, 100 mg/ml herring sperm DNA, 250mg/ml tRNA, and 60 mM dithiothreitol. For the detectionof PAC1-R mRNA, a mix of two probes was used (23 106

cpm). After overnight hybridization, slides were washed in

68 K. PEETERS ET AL.

four changes of 13 SSC at 55°C for a total of 1 hour. Slideswere dipped in water, dehydrated in an ethanol series(60%, 80%, 95%, and 100% ethanol), and air dried. Thedried hybridization sections were exposed to autoradio-graphic film (bMax Hyperfilm; Amersham Pharmacia Bio-tech, Gent, Belgium) for 17 days at room temperature.Adjacent hybridized sections were dipped in photographicemulsion (LM-1; Amersham Pharmacia Biotech) and keptin a dark box for 5 weeks. The film and sections were thendeveloped in Kodak D19 (Kodak, Belgium) for 5 minutes,fixed in rapid fixer (Ilford Hypam; Kodak) for 10 minutes,and rinsed in distilled water. The dipped sections werecounterstained with thionine, dehydrated in ethanol, andxylene and coverslipped.

Specificity controls. The specificity of the hybridiza-tion signals was confirmed in the following control exper-iments: 1) Some sections were hybridized with radioactive-labeled probes in competition with a 100-fold molar excessof the same unlabeled probes. 2) Some sections were in-cubated with the labeled sense probe instead of the respec-tive antisense oligodeoxynucleotide. 3) Some sections werepretreated with 0.005% ribonuclease A (Sigma) in 0.1 MPBS for 1 hour at 37°C. 4) Some sections were hybridizedwith a [35S]-labeled chicken VIP (chVIP) probe (59-GC-GGCTGTAGTTGTCAGTGAAGACAGCATCAGAGTGGC-GTTTGAC-39; Kuenzel et al., 1997) instead of a PACAPprobe. All control hybridization experiments were per-formed simultaneously with sections treated according tothe procedure described above.

RESULTS

ISH with the specific antisense oligodeoxynucleotideprobes revealed a widespread distribution of both PACAP-precursor and PAC1-R mRNA in the chicken brain. Thespecificity of the hybridization signal is supported by sev-eral criteria. Hybridization experiments with a chVIPprobe revealed a different distribution pattern within thebrain compared with our chicken PACAP (chPACAP)probe (data not shown). This distribution was the same asthat described by Kuenzel et al. (1997). For PAC1-R, iden-tical or fully comparable regional hybridization patternswere observed with two different oligonucleotide probesthat were derived from totally different parts of the codingsequence of the PAC1-R gene. To intensify the signal,further mapping of the brain was done with a mix of thesetwo probes. No significant hybridization was observed inparallel experiments with radioactive-labeled probes inthe presence of a 100-fold excess of the respective unla-beled probes. Also, after treatment with ribonuclease A,no labeling was observed on tissue sections taken fromseveral brain regions. For an additional control, we usedhybridized brain sections with [35S]-labeled sense oligode-oxynucleotide probes. The antisense probes were appliedto adjacent sections. Only the latter showed reaction(Fig. 1).

Distribution of PACAP mRNA

Data were organized by coronal sections taken from theanterior telencephalon to the caudal extent of the brain-stem (Figs. 2, 4). The distribution of PACAP mRNA isrepresented schematically in Figures 3 and 5. These sche-matic diagrams were made according to a published atlasof the chick brain (Kuenzel and Masson, 1988). The dia-

grams completely match the autoradiographs and indicatethe location of cells that contain PACAP mRNA.

Telencephalon. In the dorsal telencephalon, moder-ate PACAP mRNA expression was found within the hy-perstriatum accesorium (HA; Figs. 2, 3A–D). Further cau-dally, high labeling was observed in the hippocampus (Hp;Figs. 2, 3C–H, 6A). Only a very weak hybridization reac-tion was located in the hyperstriatum intercalatum supre-mum (HIS) and in the hyperstriatum dorsale and ventrale(HD and HV, respectively; Figs. 2, 3A–F). In the ventraltelencephalon, most prominent hybridization labeling wasobserved within the neostriatum intermedium (NI; Figs.2, 3C), field L (FL; Figs. 2, 3E,F), and the nucleus basalis(Figs. 2, 3A). No labeling was detected within the lobusparolfactorius or the paleostriatum augmentatum, whichis equivalent to the caudate putamen in mammals (Figs.2, 3A–D). A few scattered, labeled cells were detected inthe paleostriatum primitivum (Figs. 2, 3C). More cau-dally, in the ventrolateral part of the telencephalon,PACAP message was observed within the archistriatumanterior (AA), the archistriatum intermedium, pars vent-ralis (AIv; Figs. 2, 3E, 6C), and the archistriatum mediale(Am; Figs. 2, 3D–F). Medial to the lateral ventricle, noPACAP expression was observed within the septal region(Figs. 2, 3C,D). Laterally, a moderate signal occurred inthe area corticoidea dorsolateralis and the cortex pirifor-mis (CPi; Figs. 2, 3E–G). In the olfactory bulbs, the mitralcell layer was labeled heavily (Fig. 10E).

Diencephalon. In the preoptic area, scattered PACAPmRNA-containing cells were found in the nucleus magno-cellularis preopticus (MPO) and in the supraoptic nucleus(SON) near the tractus septomesencephalicus (TSM; Figs.2, 3C). Within the rostral hypothalamus, a moderate re-action was observed in the nucleus anterior medialis hy-pothalami (AM) and the lateral hypothalamic region(LHy; Figs. 2, 3D). More posterior, within the midhypo-thalamic region, a very strong hybridization signal oc-curred in the AM and expanded laterally to the LHy andcaudally to the nucleus ventromedialis hypothalami(VMN; Fig. 6E) and the nucleus inferioris hypothalami. Amoderate reaction was found in the paraventricular nu-cleus (PVN; Figs. 2, 3E,F, 6D).

At the level of the anterior commissure, a dense labelingreaction occurred within the bed nucleus of the pallialcommissure, a structure that borders the ventral thala-mus and the dorsal hypothalamus (Figs. 2, 3D, 6B).Within the dorsal diencephalon, a population of denselypacked, PACAP mRNA-expressing cells was observed inthe nucleus tractus septomesencephalicus (nTSM) and thenucleus dorsolateralis anterior thalami (DLA; Fig. 7A).This signal continued to the area around and within thenucleus ovoidalis (OV) and the nucleus subrotundus (Fig.7B) and more caudally to the nucleus paramedianus in-ternus thalami (Figs. 2, 3E–G, 7C). Within the pretectalarea of the thalamus, distinct populations of heavily la-beled neurons were found in the nucleus spiriformis me-dialis and the nucleus pretectalis medialis (Figs. 2, 3F,G,7D).

Mesencephalon. The distribution of PACAP mRNA-expressing cells in the mesencephalon was restrictedmainly to the stratum griseum centrale of the optic tectum(Fig. 10B). A weak-to-moderate signal was observed inand around the nucleus intercollicularis and the nucleusmesencephalicus lateralis, pars dorsalis (Figs. 2, 3H, 4,5A). At the transition from the hypothalamus into the


midbrain, scattered, moderately labeled cells appeared inthe area ventralis of Tsai lateral to the oculomotor nervesand homologous with the ventral tegmental area of mam-mals. Labeled cells also were present in the nucleus teg-menti pedunculopontinus pars compacta, which is theequivalent of the substantia nigra of mammals, and theformatio reticularis mesencephali. In the dorsal mesence-phalic tegmentum, a distinct group of PACAP mRNA-expressing cells was observed in the nucleus of Edinger-Westphal (Figs. 4, 5A).

Rhombencephalon. Within the pons, a dense clusterof PACAP mRNA expressing neurons was found nearthe nucleus lemnisci lateralis, pars intermedia, in andabout the subnuclei of the parabrachial nucleus, thelocus ceruleus, and the nucleus subceruleus ventralis(Figs. 4, 5B). A more scattered group of neurons wasdetected in the lateral and medial pontine nuclei. Fur-thermore, scattered PACAP mRNA-expressing cellswere present in the nucleus olivaris superior and thenucleus motorius nervi facialis (Figs. 4, 5C,D). A dis-tinct group of cells was observed in the nucleus angu-laris (Figs. 4, 5D). More caudally, in the medulla oblon-gata, a highly dense labeled group of cells wasvisualized in the nucleus nervi glossopharyngei, the

nucleus motorius dorsalis nervi vagi, and the nucleustractus solitarius (Figs. 4, 5E,F, 7E). The labeling wasvery conspicuous and was not separated between thenuclei. Therefore, the nuclei tended to form a singlemass as the dorsal vagal complex. Scattered labeledcells also were found throughout the reticular formation(nucleus reticularis paramedianus, nucleus reticularisparagigantocellularis lateralis, and nucleus reticularissubtrigeminalis) and in the nucleus et tractus descen-dens nervi trigemini (Figs. 4, 5C–F). In the cerebellum,Purkinje cells were labeled (Fig. 10C).

Distribution of PAC1-R mRNA

The distribution of PAC1-R mRNA is represented sche-matically in Figure 9. In general, it was found thatPAC1-R mRNA was distributed more homogeneouslythroughout the brain than PACAP mRNA. High expres-sion of PAC1-R mRNA was found in the dorsal telenceph-alon (HA, HV, and neostriatum; Figs.8, 9A–D) and in theinternal granular layer of the olfactory bulbs (Figs. 1C,10F). In the caudal direction, the hybridization signalgradually decreased in intensity. In the medial telenceph-alon, a prominent accumulation of labeled somata wasobserved in the area corresponding to the septum, the

Fig. 1. A,B: Darkfield photomicrographs of chicken brain sections(archistriatum) that were processed for in situ hybridization with[35S]-labeled pituitary adenylate cyclase-activating polypeptide(PACAP) antisense (A) and sense (B) oligonucleotide probes anddipped in photographic emulsion. Only antisense probes revealed

specific labeling (A). C,D: Autoradiographs of sagittal tissue sectionsof chicken brain processed for in situ hybridization with [35S]-labeledPACAP type I receptor (PAC1-R) oligonucleotide probes. Competitionwith a 100-fold excess of cold probes revealed no labeling (D). Scalebars 5 150 mm in B (also applies to A); 3 mm in D (also applies to C).


Fig. 2. A–H: Autoradiographs of tissue processed for in situ hybridization with a [35S]-labeled probefor PACAP. Coronal sections are shown beginning with the rostral telencephalon (A) and extending to thelevel of the median eminence from the hypothalamus (H). Scale bar 5 3 mm.

Fig. 3. A–H: Schematic representation of coronal sections depict-ing the distribution of PACAP mRNA in the chicken brain. Diagramsare matched to the autoradiographs shown in Figure 2. The stipplingrepresents areas where PACAP mRNA was dense. Schematic and

stereotaxic reference points (distance in mm from Bregma) were mod-ified from the atlas of Kuenzel and Masson (1988). For abbreviations,see list.


nucleus accumbens (Ac), and the ventral paleostriatum(PVT). Cells of the nucleus taeniae were labeled moder-ately. Only a weak signal was found in the archistriatum(Figs. 8, 9C). Moderate labeling was present in the preop-tic region (nucleus preopticus medialis, MPO, and fascic-ulus diagonalis Brocae) and in the supraoptic region (Figs.

8, 9A). Moderate-to-intense hybridization signals were ob-served throughout the entire hypothalamus and thala-mus. Numerous cells expressing PAC1-R mRNA were de-tected in the SON, PVN, nucleus periventricularishypothalami, AM, LHy, and VMN (Figs. 8, 9B–D). A pop-ulation of densely packed, PAC1-R mRNA-expressing cells

Fig. 4. A–F: Autoradiographs of tissue processed for in situ hybridization with a [35S]-labeled probefor PACAP. Coronal sections are shown beginning with the mesencephalic region at the level of theoculomotor nerve (A) and extending to the caudal rhombencephalon (F). Scale bar 5 3 mm.


was confined to the nucleus infundibularis and the nu-cleus mamillaris medialis (Figs. 8, 9E). In the thalamus,the highest level of receptor gene expression was foundwithin the nTSM, the DLA, around the OV, and in thehabenular region (Figs. 8, 9C,D). Furthermore, the gran-ular layer of the cerebellum, and layers c, h, and i of thestratum griseum et fibrosum superficiale of the optic tec-tum (Kuenzel and Masson, 1988) showed moderate-to-high PAC1-R gene expression (Figs. 8, 9F). A weak hybrid-ization signal was observed throughout the entirebrainstem (Figs. 8, 9F).

DISCUSSION

Specificity of the ISH labeling

All oligonucleotide probes were derived from specificchicken PACAP (McRory et al., 1997) or PAC1-R (Peeterset al., 1999) cDNA sequences. It has been shown that exon4 of the chPACAP/GHRH gene transcript can be splicedalternatively. Three different chicken PACAP/GHRH mR-NAs were found: a predominant long form encodingPACAP and GHRH1–46 and two shorter forms encodingPACAP and only part of the GHRH sequence, respec-

Fig. 5. A–F: Schematic representation of coronal sections depict-ing the distribution of PACAP mRNA in the chicken brain. Diagramsare matched to the autoradiographs shown in Figure 4. The stipplingrepresents areas where PACAP mRNA was dense. Schematic and



Fig. 6. Darkfield photomicrographs of chicken brain sections showing the distribution of PACAPmRNA in the hippocampus (A), bed nucleus of the pallial commissura (B), archistriatum intermediumpars ventrale (C), paraventricular nucleus (D), and nucleus ventromedialis hypothalami (E). Scale bar 5150 mm.


tively, GHRH1–43 and GHRH33–46 (McRory et al., 1997).Because our PACAP oligonucleotide probe hybridizes withsequences found only in the putative exon 5 of thechPACAP/GHRH gene, the probe detects cells expressingat least one of the three splicing variants.

In mammals, six different subtypes of the PAC1-R havebeen shown to originate from a common gene by alterna-tive splicing of the region encoding the C-terminal end ofthe third intracellularloop. These subtypes diverge fromeach other by the presence or absence of an insert. There

Fig. 7. Darkfield photomicrographs of chicken brain sectionsshowing the distribution of PACAP mRNA in the nucleus dorsolate-ralis thalami (A); in the region around the nucleus ovoidalis (OV; B);in the nucleus paramedianus internus thalami (C); in the nucleus

pretectalis medialis (PTM), nucleus spiriformis medialis (SpM; D),and nucleus motorius dorsalis nervi vagi (MnX); and in the nucleussolitarius (S; E). Scale bar 5 150 mm.


are two distinct 28-amino-acid inserts, termed hip andhop1, hop2 (a 27-amino-acid insert that lacks a serineresidue in the hop1, sequence), and two combination in-serts, hiphop1 and hiphop2. The variant without an insertwas named PAC1-Rs (short; Spengler et al., 1993; Svobodaet al., 1993; Pisegna and Wank, 1996). Until now, only twochicken PAC1-R mRNAs were found: PAC1-Rs and PAC1-Rhop1 (Peeters et al., 1999). Both PAC1-R oligonucleotideprobes detect these two subtypes of the receptor. It ispossible that our probes also detect other, not yet identi-fied chicken variants that are homologous with mamma-lian PAC1-Rhip, PAC1-hop2, or PAC1-hiphop. However,previous reverse transcriptase polymerase chain reaction

experiments revealed that the short splicing variant is thepredominant form of the receptor in the chicken brain(Peeters et al., 1999).

The specificity of the hybridization signal was confirmedby all of our control experiments. Competition with a 100-fold excess of unlabeled probes and treatment with ribonu-clease A indeed abolished all labeling. Moreover, hybridiza-tion with sense olignucleotide probes instead of therespective antisense probes revealed no specific reaction.Two PAC1-R probes that were derived from totally differentregions of the receptor cDNA sequence revealed the samehybridization pattern. Finally, we compared hybridization ofthe PACAP probe with a VIP oligonucleotide probe on adja-

Fig. 8. A–F: Autoradiographs of tissue processed for in situ hybridization with [35S]-labeled probes forPAC1-R. Coronal sections are shown beginning with the anterior hypothalamus at the level of the tractusseptomesencephalicus (TSM; A) and extending to the rhombencephalon (F). Scale bar 5 3 mm.


cent sections. The hybridization patterns of these probesclearly were different. The distribution of VIP mRNA wasthe same as that described by Kuenzel et al. (1997).

Comparison with other vertebrates andsome functional considerations

Immunocytochemical and radioimmunoassay resultshave demonstrated the existence of PACAP within nervefibers and/or cells of several mammalian tissues. Consis-tent with this broad tissue distribution, PACAP possesses

Fig. 9. A–F: Schematic representation of coronal sections depict-ing the distribution of PAC1-R mRNA in the chicken brain. Diagramsare matched to the autoradiographs shown in Figure 7. The stipplingrepresents areas where PAC1-R mRNA was dense. Schematic and


Fig. 10. A,B: Brightfield photomicrographs of chicken brain sectionsshowing the distribution of PACAP mRNA in the cerebellar cortex (A)and optic tectum (B). Arrows in A indicate labeled Purkinje cells at theborder of the granular cell layer. Hybridization signal was detectedmainly in the stratum griseum centrale of the optic tectum. C,D: Bright-field photomicrographs of chicken brain sections showing the distribu-tion of PAC1-R mRNA in the granular cell layer of the cerebellum (C) andlayer i of the optic tectum (D). Arrows in C indicate unlabeled Purkinjecells. E,F: Brightfield photomicrographs of chicken brain sections show-ing the distribution of PACAP mRNA (E) and PAC1-R mRNA (F) in theolfactory bulbs. With the PACAP oligonucleotide probe, a strong hybrid-ization signal was observed in the mitral cell layer (M). PAC1-R mRNAwas detected mainly in the internal granular layer (G). For other abbre-viations, see list. Scale bars 5 50 mm in A,C,D,F; 150 mm in B,E.


Figure 10


a variety of biologic actions (Arimura, 1998). Althoughnumerous articles have been published describing thepleiotropic activities of PACAP within several mammaliantissues, almost nothing is known about the peptide withinavian species. The results of the present ISH histochemi-cal studies have provided detailed and novel informationabout the distribution of PACAP and PAC1-R mRNA-expressing perikarya in the avian CNS. The overall dis-tribution of PACAP and PAC1-R mRNA in the avian brainis consistent with previous results obtained in mammals.

At the level of the telencephalon, PACAP and/or PAC1-RmRNA expression was found in the following limbic struc-tures: Hp, archistriatum, septum, Ac, and PVT. Part of thearchistriatum likely corresponds to the mammalian amyg-dala, and the avian Ac may be homologous with the bednucleus of the stria terminalis of mammals (Reiner et al.,1983; Butler and Hodos, 1996). Davies et al. (1997) pro-posed that the chicken archistriatum should be dividedinto two basic divisions: the limbic archistriatum, whichincludes the archistriatum posterior and extends rostrallythrough the AIv into the AA, and the nonlimbic archis-triatum, which comprises the archistriatum intermedium,pars dorsalis, and the Am and largely gives rise to specificsensory, somatosensory, and motor telencephalofugal ele-ments. According to this classification, we have foundPACAP mRNA in both the limbic archistriatum (AA andAIv) and the nonlimbic archistriatum (Am). In the septumand bed nucleus of the stria terminalis of the rat, a fairlydense network of PACAP-containing fibers was demon-strated by immunohistochemistry. The PACAP-immunopositive fibers appeared to be in contact withPACAP-immunonegative cell bodies and dendrites. Im-munoreactive perikarya and a dense fiber network, re-spectively, were detected in the lateral and central amyg-daloid nucleus of the rat (Koves et al., 1991; Piggins et al.,1996).

In addition, we found moderate-to-high expression ofPACAP mRNA in nonlimbic telencephalic areas, i.e. theHA, NI, FL, CPi, and olfactory bulbs. PAC1-R mRNA wasfound throughout the entire dorsal telencephalon (HA,HIS, HV, HD, neostriatum), except the ectostriatum.These telencephalic regions receive ascending visual, au-ditory, and somatosensory projections from nuclei in thedorsal thalamus and are likely to correspond to discreteareas of the mammalian neocortex (Karten and Shimizu,1989; Butler and Hodos, 1996). In the rat neocortex, manymoderately PAC1-R mRNA-expressing cells were distrib-uted throughout all cortical layers of the cingulate andfrontal cortices, whereas lower levels were found in othercortical areas. Layer II of the cingulate, retrosplenial, andpiriform cortices was labeled more intensely (Hashimotoet al., 1996a). In the chicken thalamus, we observed thehighest expression of PACAP and/or PAC1-R mRNA in thedorsolateral thalamus, around the nucleus ovoidalis, andin the nucleus paramedianus internus thalami. Intense-to-moderate expression of PACAP mRNA and/or PAC1-RmRNA was observed in most of the regions of the hypo-thalamus, including the supraoptic, paraventricular, andinfundibular nuclei. These nuclei have been well known toexert control influences upon the pituitary through thehypothalamohypophyseal system. Immunohistochemicalstudies have revealed PACAP-containing neurons in thehypothalamus of mammals (Koves et al., 1990, 1991; Vighet al., 1991; Piggins et al., 1996) and amphibians (Yon etal., 1992), in which a dense network of PACAP-

immunoreactive fibers surrounds the capillary loops of thehypothalamohypophyseal portal system in the externalzone of the median eminence. In fish, PACAP immuno-staining was found in nerve fibers located in the parsdistalis and neurointermediate lobe (European eel:Montero et al., 1998; goldfish: Wong et al., 1998). More-over in rat, PACAP immunoreactivity was detected in thehypophyseal portal blood (Dow et al., 1994), specific high-affinity binding sites have been characterized on anteriorpituitary cells (Gottschall et al., 1990; Masuo et al., 1991),and PACAP stimulates cAMP production in adenohy-pophyseal cell cultures (Rawlings, 1994). Taken together,these data suggest that PACAP may act as a hypophysio-tropic factor. In fact, under certain conditions, PACAP hasbeen found to induce LH, FSH, GH, and ACTH release(Rawlings, 1994). Furthermore, in mammals, there is ma-jor PACAP immunoreactivity in the magnocellular neuro-nal cell bodies of the SON and PVN, the fibers of theinternal zone of the ME, and the posterior pituitary gland.These PACAP-immunoreactive fibers generally run paral-lel within the paraventriculoinfundibular tract toward theposterior pituitary gland, similar to oxytocin and argininevasopressin neurons, suggesting a role for PACAP in pos-terior pituitary gland function in mammals (Rawlings,1994).

In addition to the regions mentioned above, PACAPmRNA and PAC1-R mRNA were found in many otherhypothalamic nuclei of the chicken. Moreover, immunocy-tochemical studies describe a dense network of PACAP-immunoreactive fibers throughout the hypothalamus(Koves et al., 1990, 1991; Vigh et al., 1991; Yon et al.,1992; Piggins et al., 1996). Because PACAP can stimulatecAMP production in hypothalamic neurons and astro-cytes, and because PACAP is a potent vasodilatator, itmay play a multifunctional role as a neurotransmitter,neuromodulator, vasoregulator, and regulator of astrocyteactivity in addition to its roles as a putative hypophysio-tropic factor and posterior pituitary hormone (Arimura,1998; Nowak et al., 1999).

In the brainstem, we found the highest expression ofPACAP mRNA in the dorsal vagal complex (which in-cludes the nucleus of the solitary tract and the dorsalmotor vagal nucleus) and the parabrachial complex. Scat-tered labeled cells were observed in the pontine nuclei, inthe ventral medulla, and throughout the reticular forma-tion. In rat, an immunohistochemical study showed thatthe majority of PACAP-positive perikarya were located inthe visceral areas of the solitary nucleus, dorsal motorvagal nucleus, nucleus ambiguus, ventrolateral medulla,ventral medullary surface, and caudal raphe nuclei (Leg-radi et al., 1994). These morphologic data support thehypothesis that PACAP is a central regulator of visceralfunctions. The nucleus of the solitary tract is a complexintegration center that subserves many diverse functions,including the processing and relaying of gustatory sensa-tion and the regulation of gastrointestinal, respiratory,and cardiovascular functions. In addition to informationarising from the periphery through the cranial nerves,higher autonomic centers, including the hypothalamus,also reach the nucleus of the solitary tract through de-scending neuronal pathways. Furthermore, the nucleus ofthe solitary tract projects directly to multiple regions ofthe CNS (pons, parabrachial nuclear complex, hypothala-mus, dorsal thalamus, limbic structures, spinal cord, etc.).The nucleus solitarius contains a great diversity of neu-


roactive substances that may act at this level as classicaltransmitters and/or neuromodulators (Jean, 1991; Butlerand Hodos, 1996).

In the chicken cerebellum, Purkinje cells were labeleddensely by the PACAP oligonucleotide probe. Granulecells were labeled moderately with the PAC1-R probes. Inadult rats, PACAP immunostaining was demonstrated inthe somata of Purkinje cells and their processes. PACAPtype I binding sites were detected on granular cerebellarcells of rats. The functional role of these findings remainsto be elucidated; however, it can be speculated thatPACAP may act as a transmitter in neuronal circuitry,sending signals from the cerebellar cortex to deep nuclei.Innervation of the granule cell layer by PACAP immuno-reactive fibers also has been observed in rat (Nielsen et al.,1998). This is in good agreement with the presence ofPACAP receptor mRNA in this cell layer in the chicken. Inrat, binding studies revealed highly specific binding siteson granule cells (Basille et al., 1994). The origin of thePACAP-immunoreactive fibers in the cerebellar granulecell layer remains to be clarified, but it is likely that theyrepresent mossy fibers projecting to the cerebellar cortexfrom the vestibular nuclei, the pontine nuclei, or the lat-eral reticular nucleus (Butler and Hodos, 1996). Indeed,we found chicken PACAP mRNA in cells of the pontinenuclei and reticular formation. Furthermore, during de-velopment, PACAP probably plays a role in the prolifera-tion, survival, and differentiation of cerebellar granulecells (Gonzalez et al., 1997; Vaudry et al., 1999).

CONCLUSIONS

Mapping the distribution of PACAP mRNA and PAC1-RmRNA within the chicken CNS has provided informationabout the sites of PACAP action in the avian brain andrepresents a critical first step toward understanding thefunction of PACAP in birds. The general regional distri-bution of PACAP and PAC1-R mRNA in the avian brain isconsistent with previous results obtained in mammals.The presence of PACAP and PAC1-R mRNA in a variety ofbrain areas in birds suggests that PACAP mediates sev-eral physiologically important processes in addition toregulating the activity of the pituitary gland.

ACKNOWLEDGMENTS

This research was supported by a grant from the QueenElisabeth Medical Foundation and by the ISERM/MVGCooperation Agreement. K.P. is supported as a ResearchAssistant by the Fund for Scientific Research—Flanders(Belgium), and H.H.J.G. is supported as a Research As-sistant by the Flemish Institute for the AdvancementScientific and Technological Research in the Industry.L.A. was supported as a postdoctoral fellow by the Fundfor Scientific Research—Flanders (Belgium).

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Distribution of pituitary adenylate cyclase-activating polypeptide and pituitary adenylate cyclase-activating polypeptide type I receptor mRNA in the chicken brain