Sites of gene expression for vasoactive intestinal polypeptide throughout the brain of the chick (Gallus domesticus)

Sites of Gene Expression for VasoactiveIntestinal Polypeptide Throughout theBrain of the Chick (Gallus domesticus)

WAYNE J. KUENZEL,1* SUSAN K. MCCUNE,2 RICHARD T. TALBOT,3

PETER J. SHARP,3 AND JOANNA M. HILL4

1Department of Poultry Science, University of Maryland, College Park, Maryland 207422Department of Pediatrics, C.M.S.C., Johns Hopkins University School of Medicine,

Baltimore, Maryland 212873Division of Development and Reproduction, Roslin Institute Edinburgh, Roslin, Midlothian,

Scotland EH25 9PS, United Kingdom4Section on Developmental and Molecular Pharmacology, Laboratory of DevelopmentalNeurobiology, National Institute of Child Health and Human Development, National

Institutes of Health, Bethesda, Maryland 20892

ABSTRACTThe peptide neurotransmitter vasoactive intestinal polypeptide (VIP) has several impor-

tant functions in vertebrates, particularly, influencing the neuroendocrine and autonomicnervous systems both in developing and in adult animals. To document potential brain areasthat might play significant functional roles, the distribution of VIP mRNA was examinedthroughout the entire chick brain by using in situ hybridization histochemistry (ISHH). Inaddition, a VIP binding-site study was completed that focused on the lateral septal organ(LSO), a circumventricular organ of potential significance in avian species. The areas whereVIP message was found included the olfactory bulbs, posterior hippocampus, parahippocam-pal area, hyperstriatum, archistriatum/nucleus (n.) taenia (amygdala), medial part of theLSO, organum vasculosum of the lamina terminalis, medial preoptic region, bed n. of thepallial commissure, anterior hypothalamic (hypo.) n., lateral hypo. area (most extensive anddensemessage), periventricular hypo. n., lateral to the paraventricular n., ventromedial hypo.n., stratum cellulare externum, inferior hypo. n., infundibular hypo. n., median eminence,three layers within the stratum griseum et fibrosum superficiale, area ventralis of Tsai, n.tegmenti pedunculopontinus pars compacta (substantia nigra), intercollicular n., central gray,locus ceruleus, parabrachial n., ventrolateral medulla, reticular pontine area, in and aboutthe n. vestibularis descendens. When compared with immunocytochemistry that detected thepresence of the peptide product VIP, more areas of the brain were found to contain perikaryaexpressing VIP by using ISHH, particularly in the telencephalon and the mesencephalon. VIPbinding sites were found in the lateral portion of the LSO where the blood-brain barrier is notfully developed. Hence, the LSO was found to contain neural elements that synthesize as wellas bind VIP. VIP appears to be a useful peptide for defining major components of the visceralforebrain system in birds. J. Comp. Neurol. 381:101–118, 1997. r 1997 Wiley-Liss, Inc.

Indexing terms: circumventricular organs; visceral forebrain system; in situ hybridization

histochemistry

Vasoactive intestinal polypeptide (VIP) is a conservedpeptide that consists of 28 amino acids. There are only fouramino acid substitutions between the compound found inbirds and that sequenced in the human (Nilsson, 1975).VIP has also been shown to have similar functions in thetwo classes of vertebrates. Specifically, it has been shownto be a prolactin (PRL)-releasing hormone in rats andmonkeys (Kato et al., 1978; Rotsztejn et al., 1980; Frawleyand Neill, 1981) and in turkey and bantam hens (Proud-man and Opel, 1983; Macnamee et al., 1986). More re-

cently, VIP has been shown to increase pituitary PRLmRNA in chickens and turkeys (Talbot et al., 1991; Xu et

Contract grant sponsor: U.S.D.A.; Contract grant number: 90-37240-5506.*Correspondence to: Dr. Wayne J. Kuenzel, Department of Animal and

Avian Sciences, University of Maryland, College Park, MD 20742.E-mail: [email protected] 8 November 1995; Revised 5 November 1996; Accepted 11

December 1996

THE JOURNAL OF COMPARATIVE NEUROLOGY 381:101–118 (1997)

r 1997 WILEY-LISS, INC.

al., 1992). A physiological consequence of PRL secretion inmammals is a lactogenic response. In the absence ofmammary glands, some birds, e.g., pigeons, produce crop-milk following increased PRL release (Peczely and Kiss,1988). A behavioral consequence of increased PRL secre-tion in some birds is an induction of incubation behavior(Sharp et al., 1989; Youngren et al., 1991). To date,intracerebroventricular infusion of PRL (Youngren et al.,1991) but not VIP (Pitts et al., 1994) has been effective ineliciting incubation behavior in turkey hens. Other func-tional similarities of VIP between the two vertebrateclasses include vasodilation, increased blood flow, exocrinegland secretion demonstrated in mammals (Shimizu andTaira, 1979; Bloom and Edwards, 1980; Heistad et al.,1980;Andersson et al., 1982), and increased secretion fromsalt glands (an exocrine gland) in birds (Gerstberger,1988). VIP has also been shown to be involved in energymetabolism in the mammalian brain (Magistretti et al.,1981; McCulloch et al., 1983) and is a key marker foridentifying the visceral forebrain system in chicks (Kuen-zel and Blahser, 1993), a system that has been proposed tooverride homeostatic mechanisms during periods of stressor emotional activity (van der Kooy et al., 1984). Inaddition, the peptide is a modulator or metabolic compo-nent of circadian rhythms in rats and is found within thesuprachiasmatic nucleus (SCN), whose activity can beinfluenced by photoperiod (Moore, 1983; Yuwiler, 1983;

Card andMoore, 1984). In birds, a subset of VIP-immunoreac-tive (ir) neurons within the medial basal hypothalamus andseptal region of the dove brain has been proposed to beencephalic photoreceptor neurons (Silver et al., 1988).Recently, chicken VIP (cVIP) cDNA clones have been

isolated and analyzed to show that the cVIP gene is similarin structure to that of mammalian VIP genes. One differ-ence is that the cVIP gene is alternatively spliced toproduce two different mRNAs. The predominant one codesfor VIP only, whereas a relatively rare one (approximately2% in hypothalamic tissue) codes for VIP and peptidehistidine isoleucine (PHI; Talbot et al., 1995). There iscurrently no evidence for alternative splicing of mamma-lian VIP mRNA (Linder et al., 1987). A second difference isthat PHI of the chicken does not have an amidation signalat its C terminus (McFarlin et al., 1995). The purpose ofour study was to map gene expression of VIP in the chickbrain and compare that distribution with past studiescompleted in the same species and sex (Kuenzel andBlahser, 1994), other avian species (Yamada et al., 1982;Mikami and Yamada, 1984; Korf and Fahrenkrug, 1984;Mikami, 1986; Peczely and Kiss, 1988; Silver et al., 1988;Mauro et al., 1989; Norgren and Silver, 1990; Aste et al.,1995; Bottjer and Alexander, 1995), and the bantam hen(Macnamee et al., 1986), where immunocytochemistry wasutilized to locate VIP-like-ir neurons. One brain structure,the lateral septal organ (LSO), which was proposed previ-

Abbreviations

AIv archistriatum intermedium, pars ventralisAM nucleus anterior medialis hypothalamiAp archistriatum posteriorAPH area parahippocampalisAVT area ventralis of TsaiBO bulbus olfactoriusCA commissura anteriorCDL area corticoidea dorsolateralisCHCS tractus corticohabenularis et corticoseptalisCPP cortex prepiriformiscsf-cn cerebrospinal fluid-contacting neuronsDSD decussatio supraoptica dorsalisE ectostriatumFLM fasciculus longitudinalis medialisFPL fasciculus prosencephali lateralis (lateral forebrain bundle)FRL formatio reticularis lateralis mesencephaliFRM formatio reticularis medialis mesencephaliGCt substantia grisea centralisGLv nucleus geniculatus lateralis, pars ventralisHA hyperstriatum accessoriumHD hyperstriatum dorsaleHIS hyperstriatum intercalatum supremumHM nucleus habenularis medialisHp hippocampusHV hyperstriatum ventraleHVd hyperstriatum ventrale, pars dorsalisHVv hyperstriatum ventrale, pars ventralisICo nucleus intercollicularisIH nucleus inferioris hypothalamiIN nucleus infundibuli hypothalamiIP nucleus interpeduncularisLA nucleus lateralis anterior thalamiLHy regio lateralis hypothalamiLLi nucleus lemnisci lateralis, pars intermediaLMD lamina medullaris dorsalisLoC locus ceruleusLSOl organum septi laterale, pars lateralisLSOm organum septi laterale, pars medialisMCC nucleus magnocellularis cochlearisMES mesencephalonMnV nucleus motorius nervi trigeminiMnVIId nucleus motorius nervi facialis, pars dorsalisMnVIIv nucleus motorius nervi facialis, pars ventralis

N neostriatumnCPa nucleus commissurae palliiNH neurohypophysisNIII nervus oculomotoriusnIX nucleus nervi glossopharyngeinPrV nucleus sensorius principalis nervi trigemininTSM nucleus tractus septomesencephalicusnX nucleus motorius dorsalis nervi vagiOA nucleus olfactorius anteriorOVLT organum vasculosum lamina terminalisPA paleostriatum augmentatumPBdl nucleus parabrachialis, pars dorsolateralisPBv nucleus parabrachialis, pars ventralisPHN nucleus periventricularis hypothalamiPME posterior median eminencePMI nucleus paramedianus internus thalamiPOP nucleus preopticus periventricularisPVN nucleus paraventricularis magnocellularisR nucleus raphesROT nucleus rotundusRpc nucleus reticularis parvocellularisRPgc nucleus reticularis pontis caudalis, pars gigantocellularisRpgl nucleus reticularis paragigantocellularisS nucleus tractus solitariusSCE stratum cellulare externumSCNm nucleus suprachiasmaticus, pars medialisSCv nucleus subceruleus ventralisSG substantia gelatinosa RolandiSGFS stratum griseum et fibrosum superficialeSM nucleus septalis medialisSN substantia nigra (nucleus tegmenti pedunculopontinus,

pars compacta)SpM nucleus spiriformis medialisTD V nucleus et tractus descendens nervi trigeminiTEL telencephalonTn nucleus taeniae (part of amygdala)TSM tractus septomesencephalicusVeD nucleus vestibularis descendensVeM nucleus vestibularis medialisVeS nucleus vestibularis superiorVIP vasoactive intestinal polypeptideVIPr vasoactive intestinal polypeptide receptorsVL ventriculus lateralis

102 W.J. KUENZEL ET AL.

ously to be a circumventricular organ in birds due to thelack of a blood-brain barrier and to the presence of VIP-irneurons that have processes extending to the surface ofthe lateral ventricle and that are presumably in contactwith the cerebrospinal fluid (Kuenzel and van Tienhoven,1982; Kuenzel and Blahser, 1994), also appeared to haveVIP binding sites based on a study completed in anotheravian species (Hof et al., 1991). Due to the potentialfunctional importance of this circumventricular organ, abinding-site study was performed to determine where VIPreceptors may reside within the LSO.

MATERIALS AND METHODS

Animals, diet, and tissue preparation

Male broiler chicks obtained fromArborAcres were usedin the study. After hatching, ten chicks were placed in acage of a Petersime battery supplied with a heat source.Chicks were fed a standard chick starter ration containing22% protein. Chicks were exposed to a constant photope-riod of 24 hours of light. At 2 weeks of age, six chicks werekilled by cervical dislocation, and their brains removedquickly from the calvarium and immersed for 10–15seconds in 2-methylbutane cooled in dry ice (230°C).Brains were then placed in dry ice and stored at 270°Cuntil they were sectioned. All brains were sectioned in thecoronal plane at 20 µm by using a cryostat, thaw mountedon autoclaved subbed slides, and refrozen.

Oligonucleotide probes

A 45-base oligonucleotide probe was developed from acVIP cDNA clone (Talbot et al., 1995). The code namefor the probe was 415Y, its origin was from exon 5, bas-es 438–389, and the actual sequence was as follows:58-GCGGCTGTAGTTGTCAGTGAAGACAGCATCAGAG-TGGCGTTTGAC-38. The oligonucleotide 415Ywas derivedfrom the nucleotide sequence encoding the first 15 aminoacids of the processed VIP peptide; therefore, the probehybridizes with sequences found only in the putative exon5 of the cVIP gene. Recent evidence has shown that thecVIP gene transcript can be alternatively spliced. Theshorter cVIPmRNAencodes for VIP only, whereas a largerform encodes both VIP and a chicken analogue of PHI 1-27in the same protein product. The relative proportions ofthe two mRNA forms in hypothalamic tissue were 97.8%VIP only and 2.2% VIP/PHI (Talbot et al., 1995). Becauseexon 5 is expressed in both of the alternative VIP tran-scripts, the probe detects VIP- and VIP/PHI-expressingneurons. The cDNA probe was end labeled by usingterminal deoxynucleotidyl transferase and [35S]a-thio-dATP (New England Nuclear, Dupont), as previouslydescribed (Young et al., 1986). Free [35S] nucleotides wereseparated from incorporated ones by passage through aSephadex G-25 spin column by centrifugation.

In situ hybridization histochemistry

Frozen sections on slides were thawed at room tempera-ture and subsequently fixed for 5 minutes in 4% formalde-hyde in phosphate-buffered saline (PBS). Slides were thenrinsed twice in PBS and prepared for hybridization aspreviously described (Young et al., 1986). Sections wereplaced in 0.25% acetic anhydride in 0.1M triethanolamine,pH 8.0, for 10 minutes. They were then dehydratedthrough a series of ethanols: 70% for 1 minute, 80% for 1

minute, 95% for 2 minutes, and 100% for 1 minute.Sections were immersed in chloroform for 5 minutesfollowed by 100% ethanol for 1 minute and 95% ethanol for1 minute. After air drying, sections were hybridized in 130µl of a solution of 50% formamide, 4 3 standard salinecitrate (SSC), 1 3 Denhardt’s reagent, and 10% dextransulfate with 500 µg/ml of salmon sperm DNA and 100 mMdithiothreitol containing 1–3 3 106 cpm of [35S]-labeledprobe overnight at 37°C. To prevent drying, parafilmcoverslips were placed over the slides. Following overnighthybridization, parafilm coverslips were floated off theslides in 1 3 SSC. Slides were washed in three changes of2 3 SSC with 50% formamide at 40°C for a total of 1 hourfollowed by two changes of 1 3 SSC at room temperaturefor a total of 1 hour. Slides were dipped in water and in 70%ethanol and were allowed to air dry. Slides within x-raycassettes were exposed to Amersham ¬Max film for 11–17days at room temperature. The film was then developed inKodak D19 for 4 minutes and fixed. After contact exposurewith ¬Max film, sections were dipped in Kodak NTB3liquid emulsion (diluted 1:1 with water) and exposed in adesiccated, light-proof chamber for 4–6 weeks at 4°C. Thedipped slides were developed in Dektol and were counter-stained with thionin to allow for visualization of cells aswell as autoradiographic grains.

Immunocytochemistry

AVIP polyclonal antibody produced in rabbits was used inthis study. It was previously validated in chicks (Kuenzel andBlahser, 1994) and was used at a dilution of 1:3,000. A brainfrom a male chick was processed, sectioned, and immuno-stained as described inKuenzel andBlahser (1994).

Receptor autoradiography

As previously described for in situ hybridization histo-chemistry (ISHH), one chick brain was prepared, sectionedat 20 µm by using a cryostat, and mounted on gelatin-coated slides. All mounted sections were then stored undervacuum at 4°C for at least 20 hours before use. Sectionswere preincubated for 30 minutes at room temperature in10 mM HEPES with 130 mM NaCl, 4.7 mM KCl, 5 mMMgCl2, 5 mM MnCl2, 1 mM EDTA, and 1% bovine serumalbumin adjusted to pH 7.4 with NaOH (Shaffer andMoody, 1986) followed by a 1-hour incubation in the samebuffer at room temperature with 1mg/ml bacitracin and 50pM [125I]-labeled VIP (Amersham) with (control sections)and without 1 µM cVIP (Peninsula) or 10 µM guanylyl-imidodiphosphate (GMP-PNP), a stable GTPanalog (Boeh-ringer Mannheim). In a previous study (Hill et al., 1992),these concentrations of VIP and GMP-PNP were found toprovide maximum inhibition of [125I]-VIP binding on brainsections. Following incubation, the slides were transferredthrough five 1-minute rinses in 400 ml of PBS, pH 7.4, onice and were rapidly dried under a stream of cold air.Sections were placed in a cassette with LKBUltrofilm for 4days. After exposure, film was developed in Kodak D-19 at20°C for 4 minutes.

RESULTS

Control procedures and presentation format

Brain sections processed with a [35S]-labeled antisenseoligonucleotide VIP probe, designated 415Y (code for exon5 of cVIP cDNA), plus a 20-fold excess of cold 415Y yielded

BRAIN DISTRIBUTION OF VIP MRNA 103

no labeling on tissue sections taken from several brainregions. Brain sections exposed solely to the labeled anti-sense oligonucleotide VIP probe showed gene expression ofVIP. For an additional control, an [35S]-labeled senseoligonucleotide VIP probe was exposed to brain sections inthe mediobasal hypothalamus that contain high numbersof VIP neurons. The labeled antisense probe 415Y wasapplied to adjacent hypothalamic sections. Only the lattershowed labeled perikarya (Fig. 1).Data were organized by coronal sections taken from the

olfactory bulbs (bulbus olfactorius; BO) to the caudal extent ofthe brainstem. To aid in the documentation of neuroanatomi-cal results, both nomenclature and schematic diagrams wereutilized froma published atlas of the chick brain (Kuenzel andMasson, 1988). The latter were modified to show whereperikarya were located that produced VIP mRNA and fororientation of photomicrographs taken at high power.

Distribution of VIP mRNA

Telencephalon

BO and dorsal telencephalon. The most rostrally lo-cated VIPmRNAwas foundwithin the BO.High concentra-

tions of message were found within the lamina of the BO,and its medial and ventral portions were clearly discerned(Figs. 2A,B, 3A,B). In addition, a few scattered neuronsshowed label in the hyperstriatum ventrale (HV), parsdorsalis (HVd), the hyperstriatum ventrale, pars ventralis(HVv), and the cortex prepiriformis (CPP). Moving cau-dally, more scattered neurons were found in the hyperstria-tum intercalatum supremum (HIS), the hyperstriatumdorsalis (HD), and the HV (Figs. 2C–G, 3C–G, 4A–D,5A–D). Distinct expression for VIP was found in thehippocampus (Hp; Figs. 2F,G, 3F,G, 4, 5, 6A, 7A) as well asin its associated parahippocampal area (APH; Figs. 4B–F,5B–F, 6A, 7A) and in area corticoidea dorsolateralis (CDL;Figs. 4F, 5F, 6A, 7A).Basal telencephalon and septal area. VIP mRNA was

found within the paleostriatum augmentatum (PA), whichis equivalent to the caudate putamen (Karten and Dubbel-dam, 1973), as shown in Figures 2G, 3G, 4A, and 5A. In theseptal area, intense expression for VIP occurred in thepars medialis of the LSO (LSOm), a circumventricularorgan found in birds and reptiles (Kuenzel and vanTienhoven, 1982; Korf and Fahrenkrug, 1984; Hirunagi et

Fig. 1. Darkfield photomicrographs of chicken brain sections pro-cessed for in situ hybridization histochemistry with [35S]-labeledprobes and dipped in photographic emulsion. A: Series of mediobasalhypothalamus showing the base of the third ventricle and the infun-dibular nucleus (IN). Use of a labeled sense oligonucleotide vasoactive

intestinal polypeptide (VIP) probe revealed no labeled neurons. B: Anadjacent section using a labeled antisense oligonucleotide VIP probe.Clusters of light grains can be seen over perikarya of neurons in theIN. Scale bar 5 100 µm.


al., 1993; Kuenzel and Blahser, 1994). The medial portionof the LSO is characterized by the presence of cerebrospinal-contacting neurons that have a bulb-like process, whichprojects into the lateral ventricle (Figs. 2E, 3E, 8). VIPmRNA was also expressed within the nucleus (n.) taeniae(Tn; Figs. 4A, 5A), the archistriatum intermedium, parsventralis (AIv; Figs. 4B, 5B), and the archistriatum poste-rior (Ap; Figs. 4F, 5F, 6A, 7A), which have all been

suggested to be equivalent to the amygdala of mammalianbrains (Zeier and Karten, 1971).Diencephalon

Preoptic area and hypothalamus. The greatest expres-sion for VIP mRNA occurred within the diencephalon.Specifically, dense accumulation of VIP mRNA was foundwithin the n. preopticus periventricularis (POP) and theorganum vasculosum of the lamina terminalis (OVLT;

Fig. 2. A–G: Autoradiographs of tissue processed for in situhybridization with a [35S]-labeled oligonucleotide probe for VIP. Coro-nal sections are shown, beginning with the olfactory bulbs (A) andextending to the level of the anterior hypothalamus (G). The finalradiographs of A, E, and F were retouched at the loci indicated by thearrowheads. Reasons for removing what appeared to be artifacts were

that all loci found were on one side of the brain only (asymmetricaldistribution), darkened loci were unsightly and distracting, and cleargroupings of grains were never found in the same brain region ofemulsion-dipped slide material that was analyzed subsequently underhigh magnification. For abbreviations, see list. Scale bar 5 1 mm.


Fig. 3. A–G: Schematic diagrams of transverse sections of chicktelencephalon matched to the autoradiographs shown in Figure 2. Thestippling represents areas where mRNA VIP was dense. The numberin each upper left hand corner shows the anterior distance in mm from

the zero coordinates given in the stereotaxic atlas of the chick brain(Kuenzel and Masson, 1988). The vertical bar to the right in eachdiagram indicates size in mm. For abbreviations, see list.


Figs. 2F, 3F), a circumventricular organ (Dellmann, 1964;Kuenzel and Blahser, 1991). The highest accumulationrecorded in any one structure was found in the lateral

hypothalamic area (LHy; Figs. 2F,G, 3F,G, 4A–D, 5A–D),with some scattered concentrations of VIP mRNA in thestratum cellulare externum (SCE; Figs. 4C, 5C), the latter

Fig. 4. A–F: Autoradiographs of tissue processed for in situ hybridization with a [35S]-labeledoligonucleotide probe for VIP. Coronal sections are shown, beginning with the midhypothalamus/caudaltelencephalic region (A) and extending to the end of the telencephalon/midmesencephalic region (F). Forabbreviations, see list. Scale bar 5 1 mm.


of which has been regarded as comparable to the postero-lateral hypothalamus of the rat (van der Kooy et al., 1984;Berk, 1987; Wild et al., 1990). It was difficult to ascertainany demarcation between the VIPmRNAfound in the LHyand the n. anterior medialis hypothalami (AM; Figs. 2G,3G), between the LHy and the SCN, pars medialis (SCNm;Figs. 2G, 3G), or between the LHy and lateral to andwithin the paraventricular hypothalamic n. (PVN; Figs.4A, 5A). In effect, a continuous group of neurons express-ing VIP mRNA was found within and about the LHy. Adistinct group of neurons displaying VIPmRNAwas found

within the n. inferioris hypothalami (IH), the n. infun-dibuli hypothalami (IN; Figs. 4C–E, 5C–E), the medianeminence (particularly its posterior portion; PME), andthe neurohypophysis (NH; Figs. 4E, 5E). A few scatteredperikarya containing VIP mRNAwere also found in the n.periventricularis hypothalami (PHN; Figs. 4D, 5D).Thalamus. A distinct expression of VIP message was

found within the n. commissurae pallii (bed n. of the pallialcommissure; nCPa), a structure that borders the ventralthalamus and the dorsal hypothalamus (Figs. 2G, 3G). VIPmRNA is continuous from the nCPa caudally to thebeginning of the habenular region. This is depicted inFigures 2G and 3G, which show a group of neuronscontaining VIPmRNA in the nCPa that continues dorsallyto the end of the n. septalis medialis (SM; Figs. 4A, 5A).The tractus corticohabenularis et corticoseptalis (CHCS)is a convenient marker that shows the point at which theseptal area separates from the thalamus and marks thebeginning of the habenular region. VIP mRNA was alsofound within the n. tractus septomesencephalicus (nTSM;Figs. 4C, 5C). Within the pretectal area of the thalamus, VIPmRNA was found in the n. spiriformis medialis (SpM; Figs.4D, 5D). ScatteredVIPmRNAwas also found in andabout then. paramedianus internus thalami (PMI; Figs. 4D, 5D).Mesencephalon

Optic tectum (colliculus mesencephali). A complex dis-tribution of VIP mRNA was found within the stratumgriseum et fibrosum superficiale (SGFS) of the optic tec-tum or colliculus mesencephali (Figs. 4C–F, 5C–F, 6A,B,7A,B). The layers of the SGFS [layers 2–12 of the numeri-cal system of Ramon y Cajal (1911) or layers a–j of thealphabetic system of Cowan et al. (1961)] that displayedVIP mRNA included 3,8, and 10–12 or b,g and i–j, respec-tively. The most dense concentrations of VIP mRNA wereconsistently in the ventralmost portion of the optic tectumin layers 10–12 (Figs. 4D–F, 5D–F, 6A, 7A). Higher magni-fication of the ISHH and immunocytochemical resultsshowed that, within layers 10–12, there were parallel rowsof neurons containing VIPmRNA (Fig. 10B), and the particu-lar neuronal types found were bipolar cells (Fig. 10C). Theneurites of all VIP-like bipolar neurons were oriented perpen-dicular to the external surface of the optic tectum.Central gray and tegmentum (substantia grisea centralis

and tegmentum mesencephali). Significant amounts ofVIPwere found in discrete groupings of neurons within themidbrain core. The area ventralis of Tsai (AVT; Figs. 4F,5F, 6A, 7A) and the n. interpeduncularis (IP; Figs. 6A, 7A)showed high levels of gene expression for VIP. Similarconcentrations of VIP mRNA were also found within thesubstantia nigra (SN; Figs. 6A, 7A), a structure that waspreviously termed the n. tegmenti pedunculopontinus,pars compacta (Karten and Dubbeldam, 1973). A discretedistribution of VIP mRNA was also found juxtapositionedto the midline of the midbrain, which then showed asymmetrical bifurcation within the central gray on bothsides of the brain (GCt; Figs. 4E,F, 5E,F, 6A, 7A). Consis-tent groupings of perikarya containing VIP mRNA werefound in the n. intercollicularis (ICo; Figs. 4D–F, 5D–F, 6A,7A), whereas a more scattered distribution of VIP geneexpression occurred throughout the formatio reticularismedialis (FRM) and lateralis (FRL) mesencephali or themedial and lateral reticular formations (Figs. 4F, 5F, 6A, 7A).Rhombencephalon

Pons, cerebellum, and medulla oblongata. Within thepons, gene expression for VIP was found in neurons

Fig. 5. A–F: Schematic diagrams of transverse sections of chicktelencephalon, diencephalon, and mesencephalon matched to theautoradiographs shown in Figure 4. The stippling represents areaswhere mRNA VIP was dense. The number in each upper left handcorner shows the anterior distance in mm from the zero coordinatesgiven in the stereotaxic atlas of the chick brain (Kuenzel and Masson,1988). The dashed line dorsal to theAp in C is the lamina archistriata-lis dorsalis. The vertical bar to the right in each diagram indicates sizein mm. For abbreviations, see list.


located in the n. parabrachialis (PB), pars ventralis (PBv)and pars dorsolateralis (PBdl; Figs. 6B, 7B), whereas morescattered neurons containing VIP mRNAwere found nearthe n. lemnisci lateralis, pars intermedia (LLi), and the n.subceruleus ventralis (SCv). VIP mRNA was also locatedin and about the locus ceruleus (LoC; Figs. 6B, 7B). Nogene expression for VIP was found in the cerebellum.Moving caudally through the pons and medulla oblongata,VIP was expressed in the ventrolateral medulla. Specifi-cally, VIP mRNA was found in and about the n. motoriusnervi facialis, pars ventralis (MnVIIv), and the n. subce-ruleus ventralis (SCv; Figs. 6C, 7C). Using the densecluster of VIP neurons in the MnVIIv and the SCv as areference point, a more scattered group of VIP mRNA-containing neurons was found oriented diagonally be-tween the MnVIIv and the n. motorius nervi facialis, parsdorsalis (MnVIId). These VIP mRNA-containing neuronswere found predominantly in the n. reticularis pontiscaudalis, pars gigantocellularis (RPgc; Figs. 6C, 7C). Morecaudally, gene expression for VIP was found in and aboutthe n. reticularis para gigantocellularis lateralis (Rpgl)

and the n. reticularis parvicellularis (Rpc; Figs. 6D, 7D).Finally, VIP mRNA-containing neurons were found in andabout the n. vestibularis descendens (VeD; Figs. 6E, 7E) andthe substantia gelatinosaRolandi (SG; Figs. 6F1,F2, 7F).

In vitro autoradiography

Results of the VIP binding study are shown in Figure 11.Note that control sections where unlabeled VIP (10–6 M)was added with the ligand showed no areas with binding(Fig. 11A). Binding sites for labeled VIP were found withinthe lateral portion of the LSO (LSOl; Fig. 11B). Thereceptors appeared to be GTP sensitive, because additionof the stable GTP analog, GMP-PNP, reduced the bindingof [125I]-VIP (Fig. 11C; Hill et al., 1992).

DISCUSSION

This study has shown a considerable, widespread distri-bution of gene expression for VIP within the chick brain.The following is a comparison of published accounts of

Fig. 6. A–F: Autoradiographs of tissue processed for in situ hybridization with a [35S]-labeledoligonucleotide probe for VIP. Coronal sections are shown, beginning with the mesencephalic region at thelevel of the oculomotor nerve (A) and extending to the caudal rhombencephalon (F). Scale bar 5 1 mm.


VIP-like neurons in which the peptide product was identi-fied in a number of avian species utilizing immunocyto-chemistry (ICC). The ICC data were compared with theVIP mRNA results reported herein using ISHH. A sum-mary is presented in Table 1, and the two sections belowwill emphasize key differences and similarities.

Key differences obtained between ICC andISHH or between avian and mammalian data

Cerebral cortex. The avian equivalent for themamma-lian cerebral cortex includes all structures dorsal to thelamina medullaris dorsalis (LMD; Fig. 3E,G) or all struc-tures within the dorsal ventricular ridge (Kallen, 1953,1962; Karten and Dubbledam, 1973; Tsai et al., 1981a,b;Veenman and Gottschaldt, 1986; Reiner et al., 1989;Dubbeldam, 1991; Karten, 1991; Rehkamper and Zilles,1991; Veenman and Reiner, 1994). Specifically, the avian

cortex includes the HA (Fig. 3E), the HIS, the HD, the HV,the neostraitum, the ectostriatum (Fig. 3C,D), and the Hpcomplex (Fig. 5). The curious findings in avian studies thatinvolve mapping cell bodies that show VIP-like-ir usingICC are the few neurons detected in the cortex (Kuenzeland Blahser, 1994;Aste et al., 1995; Bottjer andAlexander,1995). In contrast, both radioimmunoassay (Emson et al.,1979; McGregor et al., 1982; Nobou et al., 1985) and ICC(Fuxe et al., 1977; Loren et al., 1979; Morrison et al., 1984;Abrams et al., 1985) studies have shown that the cerebralcortex of mammals contains very high quantities of VIPand VIP-like neurons.The present ISHH study, however, has shown VIP

mRNA in the HV, the HIS, the HD, the neostriatum, andthe Hp (Figs. 2–5). Why the presence of message but notthe peptide in these cortical structures? Perhaps theprocessing of VIP mRNA to the VIP neural peptide does

Fig. 7. A–F: Schematic diagrams of transverse sections of chickmesencephalon and rhombencephalonmatched to the autoradiographs shown in Figure 6. The stippling represents areas where mRNAVIP wasdense. The number in each upper left hand corner shows the anterior distance in mm from the zerocoordinates given in the stereotaxic atlas of the chick brain (Kuenzel and Masson, 1988). The vertical barto the right in each diagram indicates size in mm. For abbreviations, see list.


not occur in the avian cerebral cortex. Alternatively,because the chicken is capable of producing two VIP genetranscripts, a shorter form that codes for VIP only and a

longer mRNA coding for both VIP and PHI (Talbot et al.,1995), PHI may be the peptide in this gene complex that isexpressed in some manner in the avian cortex. To our

Fig. 8. A–E: A schematic diagram of a transverse section of thechick telencephalon at the beginning of the preoptic area (A). Thenumber in the upper left hand corner shows the anterior distance inmm from the zero coordinates given in the stereotaxic atlas of thechick brain (Kuenzel and Masson, 1988). The vertical bar to the rightindicates size in mm. On the right (B) is an autoradiograph of tissueprocessed for in situ hybridization with [35S]-labeled probe for VIP.C,D: Photomicrographs of chick brain sections processed for in situhybridization with [35S]-labeled probe for VIP, dipped in photographicemulsion, and stained with thionin. Clusters of grains can be seen over

cells enriched with mRNA for VIP. The area of the brain where C wastaken is shown by the boxed area in A. D shows an enlarged view of C.The clusters of mRNA for VIP are over the perikarya of cerebrospinalfluid-contacting neurons shown in E. In E, VIP-like neurons immuno-stained with an antibody to VIP show cell bodies with a bulb-likeprojection into the base of the lateral ventricle. All cerebrospinalfluid-contacting neurons are found within the medial division of theLSO (LSOm). For abbreviations, see list. Scale bars 5 1 mm in B, 100µm in C, 40 µm in D, 30 µm in E.


knowledge, no study utilizing a specific antibody to avianPHI has reported the distribution of PHI in the telencepha-lon or in other regions of the avian brain. Another intrigu-ing possibility is that gene expression for VIP was pro-duced elsewhere and was transported to axonal andterminal fields located in these hyperstriatal and neostria-tal areas. Neuronal structures other than perikarya havebeen shown to contain mRNA for particular peptides(Jirikowski et al., 1990; Mohr and Richter, 1993). There-fore, the very finely distributed VIPmRNA in the HV, HIS,HD, and neostriatum may be a cytochemical indicator ofextraperikaryal localization of mRNA for VIP or VIP andPHI. Note, however, that denser levels of VIP mRNA(indicated by two plus signs in Table 1) were foundpredominantly above the cell body region of neurons in theHp complex (Figs. 4, 5), the amygdala (AIv, Ap, and Tn;Figs. 4A,B,F, 5A,B,F, 6A, 7A), and the caudate putamen(PA; Figs. 2D,E,G, 3D,E,G, 4A, 5A). Although no peri-karyal localization of VIP was reported in the threestructures for the chicken (Kuenzel and Blahser, 1994) orthe Japanese quail (Aste et al., 1995) utilizing ICC,VIP-like perikarya were reported in the HA (which isjuxtapositioned to the Hp) and in the PA of zebra finches(Bottjer and Alexander, 1995). In addition, unusual ir wasnoted in the piriform cortex, in its ventral extension to thearchistriatum, and in the caudal archistriatum in the

chick brain (the latter being part of the amygdala), withabundant VIP-like terminals surrounding nonreactive peri-karya (Kuenzel and Blahser, 1994). Therefore, the datasuggest that careful examination of the Hp complex,amygdala, and caudate-putamen under certain physiologi-cal states may reveal VIP-like perikarya in the three areasof the avian brain by using ICC.BO. High levels of VIP mRNA were found in the chick

BO (Figs. 2A,B, 3A,B). The result was not expected,because, to date, no reports of VIP-like perikarya withinthe BO have been reported in any avian species, yet, as canbe seen in Figure 2, VIP mRNA levels were very distinct.Curiously, VIP has been detected within the BO of chicksby using radioimmunoassay; however, it is an order ofmagnitude less than the amount found within the LHy(Kuenzel and El Halawani, unpublished data), the brainarea with the most extensive and dense concentration ofVIP neurons. In mammals, although high levels of VIPreceptors have been found in the BO (Hill et al., 1994), VIPmRNA (Hill et al., 1994) or VIP-like-ir have not beenreported; rather, VIP-like-ir has been observed in theanterior olfactory n. (Abrams et al., 1985). Due to the lowamount of VIPmRNAshown in the chick anterior olfactoryn. (Figs. 2B, 3B), the present data suggest that VIPmRNAfound within the chick BO is predominantly perikaryaland that, under certain physiological conditions, VIP-like

TABLE 1. Comparison of Perikarya Identified by Using ICC1 and ISHH2

Brain region/structure AbbreviationMammalian equivalent

(me) or English equivalent (ee)VIP-like

ir3VIP

mRNA

TelencephalonBulbus olfactorius BO Olfactory bulbs (me) None 111Hyperstriatum (subdivision) HV, HD, HIS Neocortex (me) None 1Neostriatum N Neocortex (me) None 1PaleostriatumAugmentatum PA Caudate putamen (me) None4 1 or 11Hippocampus, Area parahippocampalis Hp, APH Hippocampus (me) None5 11Archistriatum, n. Taeniae A, Tn Amygdala (me) None6 11Organum septi lateral LSO Lateral septal organ (ee) 111 111

DiencephalonN. preopticus periventricularis POP Periventricular preoptic n. (ee) None 11Organum vasculosum lamina terminalis OVLT Organum vaculosum of the lamina terminalis (me) None 11N. commissurae pallii nCPa Bed n. of the pallial commissure (ee) None 1N. anterior medialis hypo.7/n. ventromedialis hypo. AM/VMN Anterior medial hypothalamic n. (ee)/ventromedial hypothalamic n. (me) 111 111Regio lateralis hypo./n. paraventricularis hypo. LHy/PVN Lateral hypothalamic n. (me)/paraventricular hypo. n. (me) 111 111N. inferioris hypo./n. infundibuli hypo. IH/IN Inferior hypothalamic n. (ee)/arcuate n. (me) 111 111Eminentia mediana/neurohypophysis ME/NH Median eminence (me)/neurohypophysis (me) None 1N. paramedianus internus thalami/n. periventricularishypo.

PMI/PHN Internal paramedial thalamic n. (ee)/periventricular hypothalamic n. (me) None 11

N. tractus septomesencephalicus nTSM N. of septal mesencephalic tract (ee) None 1MesencephalonStratum griseum et fibrosum superficiale SGFS Superficial gray and fiber layer (ee) 1 (11)8 11 (111)8Substantia grisea centralis GCt Central gray (me) 1 1N. spiriformis medialis SpM Medial spiriform n. (ee) None 1N. intercollicularis ICo Intercollicular n. (me) 19 1Area ventralis (Tsai) AVT Ventral tegmental area (me) 1 11N. interpeduncularis IP Interpeduncular n. (me) None10 11N. tegmenti pedunculo-pontinus, pars compacta TPc Substantia nigra (me) 1 1Formatio reticularis lateralis/medialis FRL/FRM Lateral and medial reticular formation (me) None 1

RhombencephalonLocus ceruleus/ventral subceruleus LoC/SCv Locus ceruleus/ventral subceruleus (me) 1 1N. parabrachialis, pars dorsolateralis/ventralis PBdl/PBv Parabrachial complex (me) None 1N. motorius nervi facialis, pars dorsalis/ventralis MnVIId/MnVIIv Motor n. of facial nerve (me) None 1N. reticularis parvicellularis lateralis Rpc Small-celled reticular n. (ee) None 1N. reticularis paragigantocellularis lateralis Rpgl Giant-celled lateral reticular n. (ee) 1 1N. vestibularis descendens VeD Descending vestibular n. (ee) None 1Substantia gelatinosa Rolandi (Trigemini) SG Gelatinous substance (ee) None 1

1Immunocytochemistry.2In situ hybridization histochemistry.3Immunoreactivity.4VIP-like perikarya found in the PAand lobus parolfactorius of the zebra finch (Bottjer andAlexander, 1995).5VIP-like perikarya found in hyperstriatumaccessorium (HA) of zebra finches (Bottjer andAlexander, 1995). Themedioventral portion of theHAis difficult to distinguish from theHp.6Dorsal A has nonreactive perikarya surrounded by VIP-like terminals.7Hypothalami.8Ventral region of SGFS in layers 10–12 had high ir and mRNA.9VIP-like perikarya found in zebra finch (Bottjer andAlexander, 1995).10Nonreactive perikarya surrounded by VIP-like terminals.n., nucleus.


perikarya could be identified within the avian BO byapplying the procedure of ICC.Rostral lateral hypothalamic area. The area of the

chick brain with the most extensive and dense concentra-tion of VIP neurons is the LHy (Kuenzel and Blahser,1994). When one examines the mammalian diencephalon,a hypothalamic area that has a comparable density ofVIP-like neurons is the SCN (Loren et al., 1979; Card etal., 1981; Abrams et al., 1985). The SCN is regarded as theprimary circadian pacemaker within the mammalian cen-tral nervous system (Moore, 1983). A controversy haspersisted for some time regarding the location of the SCNwithin the avian brain. One site is immediately lateral tothe ventricular recess, as shown in Figure 3G (SCNm;Crosby and Woodburne, 1940; Hartwig, 1974; Bons, 1976).The second site is ventrolateral to the dorsal supraopticdecussation (DSD; Fig. 5A; Meier, 1973; Reperant, 1973;Ehrlich and Mark, 1984; Cassone and Moore, 1987; Nor-gren and Silver, 1990). There is presently no evidence ofVIP-like neurons within either of the proposed anatomicalsites of the avian SCN (Norgren and Silver, 1990). Because

VIP is such an important peptide and marker for themammalian SCN and light selectively alters VIP-ir withinthe rat SCN (Albers et al., 1987), perhaps the rostral LHy,which has such high concentrations of VIP mRNA thatoverlap with the boundaries of both the SCNm (Figs. 2G,3G) and the ventral n. of the supraoptic decussation(ventrolateral to the DSD; Fig. 9), is a component of thefunctional avian SCN responsible formediating the genera-tion of circadian rhythms. Certainly, past ablation studiesthat suggested that the SCNmregulated circadian rhythmsin birds involved electrolytic lesions, which damaged notonly the SCNm but also the rostral LHy as well (Ebiharaand Kawamura, 1981; Simpson and Follett, 1981; Takaha-shi and Menaker, 1982).Nuclei displaying discrete gene expression of VIP

and dense ir of fibers and terminals utilizing ICC.

Several nuclei and brain regions were shown to have verydistinct labeling for VIP mRNA. In addition, all had incommon a previous designation of displaying dense ir forfibers but not for perikarya (Kuenzel and Blahser, 1994).The structures included the POP and nCPa (Figs. 2F,G,

Fig. 9. A,D: Schematic diagrams of transverse sections of the chickbrain at the level of themedial and caudal hypothalamus, respectively.The number in each upper left hand corner shows the anteriordistance in mm from the zero coordinates given in the stereotaxic atlasof the chick brain (Kuenzel and Masson, 1988). The vertical bar to theright in each diagram indicates size in mm. Boxed areas show regions

where enlarged photomicrographs on the right were taken. B,C,E,F:Photomicrographs of chick brain sections processed for in situ hybrid-ization with [35S]-labeled probe for VIP, dipped in photographicemulsion, and stained with thionin. For abbreviations, see list. Scalebars 5 220 µm in B, 50 µm in C,F, 100 µm in E.


3F,G); the SM (Figs. 4A, 5A); the nTSM (Figs. 4C, 5C); thePMI and PHN (Figs. 4D,5 D); the posterior median emi-nence (PME) and neurohypophysis (NH; Figs. 4E, 5E); andthe IP and parabrachial complex (PBv and PBdl; Figs.6A,B, 7A,B). The preceding neural structures are of inter-est due to their previously described high content of VIP-irfibers and/or terminal fields. This is particularly evident inthe periventricular area of the third ventricle, the externalzone of the median eminence, the interpeduncular n., andthe parabrachial complex (Kuenzel and Blahser, 1994).The present data suggest that, when utilizing antibodiesto VIP, there is an opportunity to discover ir perikaryaunder certain physiological states. For example, an ex-tended examination of the IP was completed to establishwhether or not any ir perikarya for VIP occurred withinthe structure. The final conclusion was that what ap-peared to be some ir perikarya were actually nonreactivecell bodies that were completely surrounded by VIP-liketerminals (Fig. 5E in Kuenzel and Blahser, 1994). Thepresent study, however, suggests that, due to the intensegene expression for VIP within the IP, there appear to beboth perikarya and neurites showing mRNA for VIP.Nuclei displaying discrete gene expression of VIP

but no reported ir with ICC. Four brain structures werefound to contain VIP mRNA, but no published accounts ofeither ir perikarya or fibers utilizing ICC have beenreported. The neural structures included the OVLT (Figs.2F, 3F), the medial spiriform n. (Figs. 4D, 5D), MnVIIv,and the parvicellular portion of the reticular formation(Figs. 6C,D, 7C,D). Similar to the listed grouping ofstructures discussed previously, the present data suggestthat either VIP mRNA is synthesized but no translation tothe peptide products occur or the peptide products areproduced in too low a concentration for detection by ICC,

and only under particular physiological states will there bea possibility of identifying them.

Similarities between ICC and ISHH orbetween avian and mammalian data

LSO. Circumventricular organs (CVOs), such as theOVLT or ME, are found in most vertebrates; however, amore diverse group of CVOs has been identified in submam-malian species within the diencephalon andmedullospinalregion (Vigh and Vigh-Teichmann, 1973; Vigh-Teichmannand Vigh, 1974). Many share certain characteristics: 1)specialized ependymal cells, 2) a vascular area that has anincomplete blood-brain barrier, and 3) cerebrospinal fluid(CFS)-contacting neurons (Vigh, 1971). The latter consistof a perikaryon and a bulb-like dendritic process thatprotrudes into a ventricular space (Figs. 8E, 11D; Vigh andVigh-Teichmann, 1973; Vigh-Teichmann and Vigh, 1974).An additional CVO, termed the LSO, was proposed to existin the chick brain based on a very specialized ependymalcell layer found at the base of the lateral ventricles justlateral to the septal area (Kuenzel and van Tienhoven,1982). More recently, it has been demonstrated that thechick LSO also has an incomplete blood-brain barrier andcontains VIP-like CFS-contacting neurons (Kuenzel andBlahser, 1994). More importantly, the LSO may be a CVOfound throughout submammalian classes, because VIP-like CSF-contacting neurons have been found in thelateral septal area of Japanese quail (Yamada et al., 1982),Pekin ducks (Korf and Fahrenkrug, 1984), the ring dove(Silver et al., 1988), and a reptile (Hirunagi et al., 1993). Ofinterest is that a subpopulation of the VIP-like CSF-contacting neurons in the ring dove immunostained withan antibody against rod-opsin, which is the protein compo-

Fig. 10. A:Aschematic diagram of a transverse section of the chickbrain at the level of the mesencephalon. The number in the upper lefthand corner shows the anterior distance in mm from the zerocoordinates given in the stereotaxic atlas of the chick brain (Kuenzeland Masson, 1988). The vertical bar to the right indicates size in mm.The small box in A shows a particularly dense region of VIP mRNA,which is enlarged in B and C. B: Photomicrograph showing [35S]-labeled probe for VIP, dipped in photographic emulsion, and stained

with thionin. Dense clusters of grains over cells enriched with mRNAfor VIP are shown in layers 10–12 of the stratum griseum et fibrosumsuperficiale (SGFS).C: Immunocytochemistry of VIP-like perikarya ina layer of the SGFS taken from the same region as B. The cellulararray shows one row of bipolar neurons whose neurites are orientedperpendicular to the external surface of the optic lobe. For abbrevia-tions, see list. Scale bars 5 50 µm in B, 20 µm in C.


nent of the photopigment rhodopsin (Silver et al., 1988).Therefore, the CSF-contacting neurons of the avian LSOmight represent a component of the extraretinal ence-phalic photoreceptor involved in photoperiodic regulation(Oliver and Bayle, 1982; Foster et al., 1985). Foster et al.(1993) have recently shown that CSF-contacting neuronsin the wall of the lateral ventricle of a reptile, Anoliscarolinensis, bind antibodies raised against opsin purifiedfrom chicken cones and may correspond to the VIP-like-irCSF-contacting neurons of birds. Our data show that theCSF-contacting neurons in the chick brain not only bind toan antibody against VIP but also display gene expressionfor the peptide due to the presence of VIP mRNA in the

perikarya of neurons within the medial portion of the LSO,which corresponds to the cell bodies of CSF-contactingneurons (Fig. 8). In addition, the lateral part of the LSOcontains VIP receptors (Fig. 11). In summary, the LSO ofthe chick consists of a medial portion (LSOm), containing anumber of VIP-producing, CSF-contacting neurons, and alateral component (LSOl). The LSOl comprises the thick-ened layer of ependymal cells, an incomplete blood-brainbarrier, and VIP binding sites. Hence, to date, the LSO is astructurally complex organ in the chick, a potential CVOamong submammalian vertebrates, and one that mayserve basic functions perhaps associated with encephalicphotoreception.

Fig. 11. A–C: Autoradiographs of [125I]-VIP binding in transversesections of the chick brain at the level of the telencephalon shown inFigure 3E. A: A control section treated with [125I]-VIP in the presenceof 1 µMVIP. B:Asection showing the total [125I]-VIP binding pattern inthe ventral region of the telencephalon. Note the dense binding in theLSOl. C: A section showing [125I]-VIP binding in the presence of 10 µMguanylyl-imidodiphosphate (GMP-PNP). Binding of VIP in the LSOl ispresent but is reduced compared with B. D: A three-dimensional

composite diagram of the LSO modified from Kuenzel and Blahser(1994). The LSOm consists of the VIP-like cerebrospinal fluid-contacting neurons (csf-cn), an ependymal layer, and a thin band oftissue within the lateral septal area. The LSOl consists of a thickened,specialized ependymal layer, a dense vascular area, an incompleteblood-brain barrier, and VIP receptors (VIPr). For other abbreviations,see list. Scale bar 5 1 mm inA (also applies to B,C).


LHy-AM-VMN-PVN complex. The largest area of thebrain reported in the literature that displayed a highconcentration of VIP-like-ir perikarya was in and aboutthe LHy, including encroachment upon at least three otherhypothalamic nuclei: AM, VMN, and PVN (Kuenzel andBlahser, 1994). There was a high degree of similaritybetween the distribution of ir perikarya and the distribu-tion of VIP mRNA within this region (Figs. 2F,G, 3F,G,4A–D, 5A–D).IN/IH complex. The IN/IH complex of neurons express-

ing VIP mRNA is distinct from the preceding, much largergroup of neurons. Similar to the LHy-AM-VMN-PVNcomplex, the density of immunostaining for VIP reportedin this area (Yamada et al., 1982; Mikami and Yamada,1984; Macnamee et al., 1986; Mikami, 1986; Peczely andKiss, 1988; Silver et al., 1988; Mauro et al., 1989; Kuenzeland Blahser, 1994) correlates well with gene expression forVIP (Figs. 4C–E, 5C–E, 9D–F). It is this group of neuronsthat has been most associated with the regulation of PRLsecretion due to its proximity to the median eminence andthe reports showing that VIP stimulates release of PRLfrom the pituitary in vitro (Proudman and Opel, 1983;Macnamee et al., 1986) and in vivo (Macnamee et al., 1986)and is associated with incubation behavior (Mauro et al.,1989; Sharp et al., 1989; El Halawani and Rozenboim,1993).SGFS. A distinct layering of VIP mRNA occurred

throughout the SGFS, which included layers 2–12 of theoptic tectum or tectum mesencephali. Specific layers thatshowed gene expression were 3, 8, and 10–12 (Figs. 4C–F,5C–F, 6A, 7A, 10). The greatest density of VIP mRNAoccurred in layers 10–12, particularly in the ventralmostportion of layers 10–12 (Fig. 10A, boxed area). Of interestis that the densest staining of VIP-like-ir perikarya re-ported within the SGFS utilizing ICC occurred in the sameventralmost portion of layers 10–12 (Kuenzel and Blahser,1994). The VIP-like-ir neurons found in layers 10–12 werebipolar (Fig. 10C) and were oriented perpendicular to theexternal surface of the tectum mesencephali. The highassociation between dense ir for VIP and dense levels ofVIPmRNAwithin the same bipolar neurons in the ventral-most layers of the SGFS suggests an important functionfor layers 10–12 of the ventral SGFS (vSGFS). In particu-lar, the retinotopic organization of retinotectal projectionshas been determined for birds, showing that the vSGFSreceives projections from the dorsal retina (Hamdi andWhitteridge, 1954). Therefore, VIP neurons identified inthe vSGFS may play a key role within the visual systemassociated with the ventral visual field.AVT, GCt, ICo, SN, LoC, and SCv. Four nuclei within

the mesencephalon (AVT, GCt, ICo, and SN) and two areaswithin the caudal mesencephalic area and pons (LoC andSCv) showed discrete quantities of VIP mRNA (Figs. 4E,F,5E,F, 6A,B, 7A,B). The six nuclei also displayed VIP-like-irperikarya (Table 1).Rpgl. Anucleuswithin the rhombencephalon, the Rpgl,

showed VIP mRNA in and about the structure (Figs. 6D,7D) as well as VIP-like-ir perikarya (Table 1).

Visceral forebrain system: Comparisonbetween birds and mammals

The concept of a visceral forebrain system in mammalswas introduced by van der Kooy et al. (1984). Its functionwas to influence cardiovascular, respiratory, and gastroin-testinal functions, including the possibility of interfering

with or overriding brainstem homeostatic mechanismsduring periods of stress or emotional activity. The keyneuroanatomical components in the visceral forebrainsystem of the rat included the medial and lateral prefron-tal cortex, the central n. of the amygdala, the bed n. of thestria terminalis, the paraventricular n., the arcuate n., theposterolateral hypo. n., the parabrachial n., and the n.tractus solitarius (van der Kooy et al., 1984). An anatomi-cal requirement for the system was the demonstration of adirect neural connection among all components to eitherthe nTS or the PB (van der Kooy et al., 1984). Do avianspecies have a comparable visceral forebrain system?Based on physiological, behavioral, and anatomical stud-ies, it has been suggested that an equivalent system existswithin birds (Kuenzel and Blahser, 1993). The avianventral telencephalic and diencephalic components (aviannAc/nST, PVN, and LHy/SCE), as noted by Wild et al.(1990), best satisfy the anatomical requirements of mono-synaptic connections between each of them and the nTS orPB. Two controversial neural areas include avian struc-tures that are comparable to the mammalian amygdalaand prefrontal cortex. Recent retrograde and anterogradepathway-tracing evidence (Veeman et al., 1995) supportsan early idea that the medial and caudal portions of theavian archistriatum (n. taeniae, archistriatum mediale,and caudal complex of the archistiatum) are homologous tothe amygdala (Zeier andKarten, 1971). Of interest was thesuggestion that VIP-ir is a useful marker for the identifica-tion of components of the visceral forebrain system (Kuen-zel and Blahser, 1993; 1994). VIP mRNA, as shown inFigures 4A,B and 5A,B, was likewise identified within themedial and ventral regions of the archistriatum.The homologous structures in the avian brain for the

prefrontal cortex remain most controversial. It has beenproposed that the caudodorsolateral neostriatum is theequivalent avian structure (Mogensen and Divac, 1982;Divac and Mogensen, 1985; Divac et al., 1985; Rehkamperand Zilles, 1991; Waldmann and Gunturkun, 1993); how-ever, a monsynaptic connection to the nTS has not beendemonstrated to date (Wild et al., 1990). Based on pub-lished VIP-ir results (Kuenzel and Blahser, 1994; Bottjerand Alexander, 1995) and on the VIP mRNA resultspresented herein, the forebrain areas that appear to beappropriate for inclusion within an avian visceral fore-brain system are the BO, the Hp complex, restrictedportions of the rostral hyerstriatum (HV, HD, and HIS),and the piriform cortex. Specific neuroanatomical andfunctional studies will need to be completed to establishwhich of the latter should be the telencephalic avianequivalents of the mammalian medial and lateral prefron-tal cortex.

ACKNOWLEDGMENTS

The authors thank Manju Masson for her expert techni-cal assistance with the line drawings and photographicprints and Sheila Brown for typing the paper. This study isScientific Article A7839, Contribution 9167 of the Mary-land Agricultural Experiment Station (Department ofPoultry Science).

LITERATURE CITED

Abrams, G.M., G. Nilaver, and E.A. Zimmerman (1985) VIP-containingneurons. In A. Bjorklund and T. Hokfelt (eds.): Handbook of Chemical


Neuroanatomy, Vol. 4: GABA and Neuropeptides in the CNS, Part I.Amsterdam: Elsevier, pp. 335–354.

Albers, H.E., N. Minamitani, E. Stopa, and C.F. Ferris (1987) Lightselectively alters vasoactive intestinal peptide and peptide histidineisoleucine immunoreactivity within the rat suprachiasmatic nucleus.Brain Res. 437:189–192.

Andersson, P.-O., S.R. Bloom,A.V. Edwards, and J. Jarhult (1982) Effects ofstimulation of the chorda tympani in bursts on submaxillary responsesin the cat. J. Physiol. (London) 322:469–483.

Aste, N., C. Viglietti-Panzica, A. Fasolo, and G.C. Panzica (1995) Mappingof neurochemical markers in quail central nervous system: VIP- andSP-like immunoreactivity. J. Chem. Neurol. 8:87–102.

Berk, M.L. (1987) Projections of the lateral hypothalamus and bed nucleusof the stria terminalis to the dorsal vagal complex in the pigeon. J.Comp. Neurol. 260:140–156.

Bloom, S.R., and A.V. Edwards (1980) Vasoactive intestinal peptide inrelation to atropine resistant vasodilation in the submaxillary gland ofthe cat. J. Physiol. 300:41–53.

Bons, N. (1976) Retinohypothalamic pathway in the duck (Anas platyrhyn-chos). Cell Tissue Res. 168:343–360.

Bottjer, S.W., and G. Alexander (1995) Localization of metenkephalin andvasoactive intestinal polypeptide in the brains of male zebra finches.Brain Behav. Evol. 45:153–177.

Card, J.P., and R.Y. Moore (1984) The suprachiasmatic nucleus of thegolden hamster: Immunohistochemical analysis of cell and fiber distri-bution. Neuroscience 13:415–431.

Card, J.P., N. Brecha, H. Karten, and R.Y. Moore (1981) Immunocytochemi-cal localization of vasoactive intestinal polypeptide-containing cells andprocesses in the suprachiasmatic nucleus of the rat. Light and electronmicroscopic analysis. J. Neurosci. 1:1289–1303.

Cassone, V.M., and R.Y. Moore (1987) The retinohypothalamic projectionand suprachiasmatic nucleus of the house sparrow, Passer domesticus.J. Comp. Neurol. 266:171–182.

Cowan, W.M., L. Adamson, and T.P.S. Powell (1961) An experimental studyof the avian visual system. J. Anat. 95:545–563.

Crosby, E.C., and R.T. Woodburne (1940) The comparative anatomy of thepreoptic area and the hypothalamus. Res. Publ. Assoc. Res. Nerv.Mental Dis. 20:52–169.

Dellmann, H.-D. von (1964) Zur Struktur des Organon vasculosum laminaeterminalis des Huhnes. Anat. Anz. 115:174–183.

Divac, I., and J. Mogensen (1985) The prefrontal ‘‘cortex’’ in the pigeon:Catecholamine histofluorescence. Neuroscience 15:677–682.

Divac, I., J. Mogensen, and A. Bjorklund (1985) The prefrontal ‘‘cortex’’ inthe pigeon. Biochemical evidence. Brain Res. 332:365–368.

Ebihara, S., and H. Kawamura (1981) The role of the pineal organ and thesuprachiasmatic nucleus in the control of circadian locomotor rhythmsin the Java sparrow, Padda oryzivora. J. Comp. Physiol. 141:207–214.

Ehrlich, D., and R.Mark (1984)An atlas of the primary visual projections inthe brain of the chickGallus gallus. J. Comp. Neurol. 223:592–610.

El Halawani, M.E., and I. Rozenboim (1993) The ontogeny and control ofincubation behavior in turkeys. Poultry Sci. 72:906–911.

Emson, P.C., R.F.T. Gilbert, I. Loren, J. Fahrenkrug, F. Sundler, and O.B.Schaffalitzky de Muckadel (1979) Development of vasoactive intestinalpolypeptide (VIP) containing neurones in the rat brain. Brain Res.177:437–444.

Foster, R.G., B.K. Follett, and J.N. Lythgoe (1985) Rhodopsin-like photosen-sitivity of extra-retinal photoreceptors mediating the photoperiodicresponse in quail. Nature 313:50–52.

Foster, R.G., J.M. Garcia-Fernandez, I. Provencio, and W.J. De Grip (1993)Opsin localization and chromophore retinoids identified within thebasal brain of the lizard Anolis carolinensis. J. Comp. Physiol. A172:33–45.

Frawley, L.S., and J.D. Neill (1981) Stimulation of prolactin secretion inrhesus monkeys by vasoactive intestinal polypeptide. Neuroendocrinol-ogy 33:79–83.

Fuxe, K., T. Hokfelt, S.I. Said, and V. Mutt (1977) Vasoactive intestinalpolypeptide and the nervous system: Immunohistochemical evidencefor localization in central and peripheral neurons, particularly intracor-tical neurons in the cerebral cortex. Neurosci. Lett. 5:241–246.

Gerstberger, R. (1988) Functional vasoactive intestinal polypeptide (VIP)system in salt glands of the Pekin duck. Cell Tissue Res. 252:39–48.

Hamdi, F.A., and D. Whitteridge (1954) The representation of the retina onthe optic tectum of the pigeon. Q. J. Exp. Physiol. 39:111–119.

Hartwig, H.G. (1974) Electron microscopic evidence for a retinohypotha-lamic projection to the suprachiasmatic nucleus of Passer domesticus.Cell Tissue Res. 153:89–99.

Heistad, D.D., M.L. Marcus, S.I. Said, and P.M. Gross (1980) Effect ofacetylcholine and vasoactive intestinal peptide on cerebral blood flow.Am. J. Physiol. 239:H73–H80.

Hill, J.M., A. Harris, and D.I. Hilton-Clarke (1992) Regional distribution ofguanine nucleotide-sensitive and guanine nucleotide-insensitive vaso-active intestinal peptide receptors in rat brain. Neuroscience 48:925–932.

Hill, J.M., D.V. Agoston, P. Gressens, and S.K. McCune (1994) Distributionof VIP mRNA and two distinct VIP binding sites in the developing ratbrain: Relation to ontogenic events. J. Comp. Neurol. 342:186–205.

Hirunagi, K., E. Rommel, A. Oksche, and H.-W. Korf (1993) Vasoactiveintestinal peptide-immunoreactive cerebrospinal fluid-contacting neu-rons in the reptilian lateral septum/nucleus accumbens. Cell TissueRes. 274:79–90.

Hof, P.R., M.M. Dietl, Y. Charnay, J.-L. Martin, C. Bouras, J.M. Palacios,and P.J. Magistretti (1991) Vasoactive intestinal peptide binding sitesand fibers in the brain of the pigeonColumba livia:An autoradiographicand immunohistochemical study. J. Comp. Neurol. 305:393–411.

Jirikowski, G.F., P.P. Sanna, and F.E. Bloom (1990) mRNA coding foroxytocin is present in axons of the hypothalamo-neurohypophysialtract. Proc. Natl. Acad. Sci. USA 87:7400–7404.

Kallen, B. (1953) On the nuclear differentiation during ontogenesis of theavian forebrain and some notes on the amniote strio-amygdaloidcomplex. ActaAnat. 17:72–84.

Kallen, B. (1962) Embryogenesis of brain nuclei in the chick telencephalon.Ergebn. Anat. Entwick. Gesch. 36:62–82.

Karten, H.J. (1991) Homology and evolutionary origins of the ‘‘neocortex.’’Brain Behav. Evol. 38:264–272.

Karten, H.J., and J.L. Dubbeldam (1973) The organization and projectionsof the paleostriatal complex in the pigeon (Columba livia). J. Comp.Neurol. 148:61–90.

Kato, Y., Y. Iwasaki, J. Iwasaki, H. Abe, N. Yanaihara, and H. Imura (1978)Prolactin release by vasoactive intestinal peptide in rats. Endocrinology103:554–558.

Korf, H.W., and J. Fahrenkrug (1984) Ependymal and neuronal specializa-tions in the lateral ventricle of the Pekin duck,Anas platyrhynchos. CellTissue Res. 236:217–227.

Kuenzel, W.J., and S. Blahser (1991) The distribution of gonadotropin-releasing hormone (GnRH) neurons and fibers throughout the chickbrain (Gallus domesticus). Cell Tissue Res. 264:481–495.

Kuenzel, W.J., and S. Blahser (1993) The visceral forebrain system in birds:Its proposed anatomical components and functions. Poultry Sci. Rev.5:29–36.

Kuenzel, W.J., and S. Blahser (1994) Vasoactive intestinal polypeptide(VIP)-containing neurons: Distribution throughout the brain of thechick (Gallus domesticus) with focus upon the lateral septal organ. CellTissue Res. 275:91–107.

Kuenzel, W.J., and M. Masson (1988) AStereotaxic Atlas of the Brain of theChick (Gallus domesticus). Baltimore, MD; Johns Hopkins UniversityPress.

Kuenzel, W.J., and A. van Tienhoven (1982) Nomenclature and location ofavian hypothalamic nuclei and associated circumventricular organs. J.Comp. Neurol. 206:293–313.

Linder, S., T. Barkhem, A. Norberg, H. Persson, M. Schalling, T. Hokfelt,and G. Magnusson (1987) Structure and expression of the gene encod-ing the vasoactive intestinal peptide precursor. Proc. Natl. Acad. Sci.USA 84:605–609.

Loren, I., P.C. Emson, J. Fahrenkrug, A. Bjorklund, J. Alumets, R.Håkanson, and F. Sundler (1979) Distribution of vasoactive intestinalpolypeptide in the rat and mouse brain. Neuroscience 4:1953–1976.

Macnamee, M.C., P.J. Sharp, R.W. Lea, R.J. Sterling, and S. Harvey (1986)Evidence that vasoactive intestinal polypeptide is a physiologicalprolactin-releasing factor in the bantam hen. Gen. Comp. Endocrinol.62:470–478.

Magistretti, P.J., J.H. Morrison, W.J. Shoemaker, V. Sapin, and F.E. Bloom(1981) Vasoactive intestinal polypeptide induces glycogenolysis inmouse cortical slices: A possible regulatory mechanism for the localcontrol of energy metabolism. Proc. Natl. Acad. Sci. USA 78:6535–6539.

Mauro, L.J., R.P. Elde, O.M. Youngren, R.E. Phillips, andM.E. El Halawani(1989) Alterations in hypothalamic vasoactive intestinal peptide-likeimmunoreactivity are associated with reproduction and prolactin re-lease in the female turkey. Endocrinology 125:1795–1804.


McCulloch, J., P.A.T. Kelly, R. Uddman, and L. Edvinsson (1983) Functionalrole of vasoactive intestinal polypeptide in the caudate nucleus: A2-deoxy (14C) glucose investigation. Proc. Natl. Acad. Sci. USA 80:1472–1476.

McFarlin, D.R., D.A. Lehn, S.M.Moran, M.J. MacDonald, andM.L. Epstein(1995) Sequence of a cDNA encoding chicken vasoactive intestinalpeptide (VIP). Gene 154:211–213.

McGregor, G.P., P.L. Woodhams, D.J. O’Shaughnessy, M.A. Ghatei, J.M.Polak, and S.R. Bloom (1982) Developmental changes in bombesin,substance P, somatostatin and vasoactive intestinal polypeptide in therat brain. Neurosci. Lett 28:21–27.

Meier, R.E. (1973) Autoradiographic evidence for a direct retino-hypotha-lamic projection in the avian brain. Brain Res. 53:417–421.

Mikami, S. (1986) Immunocytochemistry of the avian hypothalamus andadenohypophysis. Int. Rev. Cytol. 103:189–248.

Mikami, S., and S. Yamada (1984) Immunohistochemistry of the avianhypothalamus and adenohypophysis. Int. Rev. Cytol. 103:189–248.

Mogensen, J., and I. Divac (1982) The prefrontal ‘‘cortex’’ in the pigeon.Behavioral evidence. Brain Behav. Evol. 21:60–66.

Mohr, E., and D. Richter (1993) Hypothalamic neuropeptide genes: Aspectsof evolution, expression and subcellular mRNA distribution. Ann. N.Y.Acad. Sci. 689:50–58.

Moore, R.Y. (1983) Organization and function of a central nervous systemcircadian oscillator: The suprachiasmatic hypothalamic nucleus. Fed.Proc. 42:2783–2789.

Morrison, J.H., P.J. Magistretti, R. Benoit, and F.E. Bloom (1984) Thedistribution and morphological characteristics of the intracortical VIP-positive cell: An immunohistochemical analysis. Brain Res. 292:269–282.

Nilsson, A. (1975) Structure of the vasoactive intestinal octacosapeptidefrom chicken intestine. The amino acid sequence. FEBS Lett. 60:322–326.

Nobou, F., J. Besson, W. Rostene, and G. Rosselin (1985) Ontogeny ofvasoactive intestinal peptide and somatostatin in different structures ofthe rat brain: Effects of hypo and hypercorticism. Dev. Brain Res.20:296–301.

Norgren, R.B., and R. Silver (1990) Distribution of vasoactive intestinalpeptide-like and neurophysin-like immunoreactive neurons and acetyl-cholinesterase staining in the ring dove hypothalamus with emphasison the question of an avian suprachiasmatic nucleus. Cell Tissue Res.259:331–339.

Oliver, J., and J.D. Bayle (1982) Brain photoreceptors for the photoinducedtesticular response in birds. Experientia 38:1021–1029.

Peczely, P., and J.Z. Kiss (1988) Immunoreactivity to vasoactive intestinalpolypeptide (VIP) and thyreotropin-releasing hormone (TRH) in hypo-thalamic neurons of the domesticated pigeon (Columba livia). Alter-ations following lactation and exposure to cold. Cell Tissue Res.251:485–494.

Pitts, G.R., O.M. Youngren, J.L. Silsby, I. Rozenboim, Y. Chaiseha, R.E.Phillips, D.N. Foster, and M.E. El Halawani (1994) Role of vasoactiveintestinal peptide in the control of prolactin-induced turkey incubationbehavior. II. Chronic infusion of vasoactive intestinal peptide. Biol.Reprod. 50:1350–1356.

Proudman, J.A., and H. Opel (1983) Stimulation of prolactin and growthhormone secretion from turkey pituitary cells. Poultry Sci. 62:1484–1485.

Ramon y Cajal, S. (1911) Histologie du Systeme Nerveux de l’Homme et desVertebres. Paris: A. Maloine.

Rehkamper, G., and K. Zilles (1991) Parallel evolution in mammalian andavian brains: Comparative cytoarchitectonic and cytochemical analy-sis. Cell Tissue Res. 263:3–28.

Reiner,A., S.E. Brauth, C.A. Kitt, and R. Quirion (1989) Distribution of mu,delta, and kappa opiate receptor types in the forebrain and midbrain ofpigeons. J. Comp. Neurol. 280:359–382.

Reperant, J. (1973) Nouvelles donnees sur les projections visuelles chez lepigeon (Columba livia). J. Hirnforsch 14:151–187.

Rotsztejn, W.H., L. Benoist, J. Besson, G. Beraud, C. Kordon, G. Rosselin,and J. Duval (1980) Effect of vasoactive intestinal peptide (VIP) on therelease of various adenohypophysial hormones from purified cellsobtained by unit gravity sedimentation. Inhibition by dexamethasone ofVIP-induced prolactin release. Neuroendocrinology 3:282–286.

Shaffer, M.M., and T.W. Moody (1986) Autoradiographic visualization ofCNS receptors for vasoactive intestinal peptide. Peptides 7:283–288.

Sharp, P.J., R.J. Sterling, R.T. Talbot, and N.S. Huskisson (1989) The role ofhypothalamic vasoactive intestinal polypeptide in the maintenance ofprolactin secretion in incubating bantam hens: Observations using

passive immunization, radioimmunoassay and immunohistochemistry.J. Endocrinol. 122:5–13.

Shimizu, T., and N. Taira (1979) Assessment of the effects of vasoactiveintestinal peptide (VIP) on blood flow through and salivation of the dogsalivary gland in comparison with those of secretin, glucagon andacetylcholine. Br. J. Pharmacol. 65:683–687.

Silver, R.P., P. Witkovsky, P. Horvath, V. Alones, C.J. Barnstable, and M.N.Lehman (1988) Coexpression of opsin- and VIP-like-immunoreactivityin CSF-contacting neurons of the avian brain. Cell Tissue Res. 253:189–198.

Simpson, S.M., and B.K. Follett (1981) Pineal and hypothalamic pacemak-ers: Their role in regulating circadian rhythmicity in Japanese quail. J.Comp. Physiol. 144:381–389.

Takahashi, J.S., and M. Menaker (1982) Role of the suprachiasmatic nucleiin the circadian system of the house sparrow, Passer domesticus. J.Neurosci. 2:815–828.

Talbot, R.T., M.C. Hanks, R.J. Sterling, H.M. Sang, and P.J. Sharp (1991)Pituitary prolactin messenger ribonucleic acid levels in incubating andlaying hens: Effects of manipulating plasma levels of vasoactive intesti-nal polypeptide. Endocrinology 129:496–502.

Talbot, R.T., I.C. Dunn, P.W. Wilson, H.M. Sang, and P.J. Sharp (1995)Evidence for alternative splicing of the chicken vasoactive intestinalpolypeptide gene transcript. J. Mol. Endocrinol. 15:81–91.

Tsai, H.M., B.B. Garber, and L.M.H. Larramendi (1981a) 3H-thymidineautoradiographic analysis of telencephalic histogenesis in the chickembryo: I. Neuronal birth dates of telencephalic compartments in situ.J. Comp. Neurol. 198:275–292.

Tsai, H.M., B.B. Garber, and L.M.H. Larramendi (1981b) 3H-thymidineautoradiographic analysis of telencephalic histogenesis in the chickembryo: II. Dynamics of neuronal migration, displacement, and aggre-gation. J. Comp. Neurol. 198:293–306.

van der Kooy, D., L.Y. Koda, J.F. McGinty, C.R. Gerfen, and F.E. Bloom(1984) The organization of projections from the cortex, amygdala, andhypothalamus to the nucleus of the solitary tract in rat. J. Comp.Neurol. 224:1–24.

Veenman, C.L., and K.-M. Gottschaldt (1986) The nucleus basalis-neostriatum complex in the goose (Anser anser L.). In F. Beck, W. Hild,W. Kriz, R. Ortmann, J.E. Pauly, and T.H. Schiebler (eds): Advances inAnatomy, Embryology, and Cell Biology. Vol. 96, Berlin: Springer.

Veenman, C.L., and A. Reiner (1994) The distribution of GABA-containingperikarya, fibers, and terminals in the forebrain and midbrain ofpigeons, with particular reference to the basal ganglia and its projectiontargets. J. Comp. Neurol. 339:209–250.

Veenman, C.L., J.M. Wild, and A. Reiner (1995) Organization of the avian‘‘corticostriatal’’ projection system: A retrograde and anterograde path-way tracing study in pigeons. J. Comp. Neurol. 354:87–126.

Vigh, B. (1973) Das Paraventrikularorgan und das ZirkumventrikulareSystem. Studia Biological Hungarica, Vol. X. Budapest: Acad Kiado.

Vigh, B., and I. Vigh-Teichmann (1973) Comparative ultrastructure ofCSF-contacting neurons. Int. Rev. Cytol. 35:189–251.

Vigh-Teichmann, I., and B. Vigh (1974) The infundibular cerebrospinalfluid-contacting neurons. Adv. Anat. Embryol. Cell Biol. 50:1–91.

Waldmann, C., and O. Gunturkun (1993) The dopaminergic innervation ofthe pigeon caudolateral forebrain: Immunocytochemical evidence for a‘‘prefrontal cortex’’ in birds? Brain Res. 600:225–234.

Wild, J.M., J.J.A. Arends and H.P. Zeigler (1990) Projections of theparabrachial nucleus in the pigeon (Columba livia). J. Comp Neurol.293:499–523.

Xu, M., J. Proudman, and M.E. El Halawani (1992) Effect of selective drugsfor dopaminergic D1 and D2 receptors on cVIP-induced PRL mRNA inturkey hens. Poultry Sci. 1(Suppl):21.

Yamada S., S. Mikami, and N. Yanaihara (1982) Immunohistochemicallocalization of vasoactive intestinal polypeptide (VIP)-containing neu-rons in the hypothalamus of the Japanese quail, Coturnix coturnix. CellTissue Res. 266:13–26.

Young, W.S., III, E. Mezey, and R.E. Siegel (1986) Vasopressin and oxytocinmRNAs in adrenalectomized and Brattleboro rats: Analysis by in situhybridization histochemistry. Mol. Brain Res. 1:231–241.

Youngren, O.M., M.E. El Halawani, J.L. Silsby, and R.E. Phillips (1991)Intracranial prolactin perfusion induces incubation behavior in turkeyhens. Biol. Reprod. 44:425–431.

Yuwiler, A. (1983) Light and agonists alter pineal N-acetyl-transferaseinduction by vasoactive intestinal polypeptide. Science 220:1082–1083.

Zeier, H., and H.J. Karten (1971) The archistriatum of the pigeon:Organization of afferent and efferent connections. Brain Res. 31:313–326.


Documents

Sites of gene expression for vasoactive intestinal polypeptide throughout the brain of the chick (Gallus domesticus)