THE JOURNAL OF COMPARATNE NEUROLOGY 305:393-411 (1991)

Vasoactive Intestinal Peptide Binding Sites and Fibers in the Brain of the Pigeon Columba Ziuia: An Autoradiographic and Immunohistochemical Study PATRICK R. HOF, MONIKA M. DIETL, YVES CHARNAY, JEAN-LUC MARTIN, CONSTANTIN BOURAS, JOSE M. PALACIOS, AND PIERRE J. MAGISTRETTI Fishberg Research Center for Neurobiology and Department of Geriatrics and Adult Development, Mount Sinai School of Medicine, New York, New York 10029 (P.R.H.); INSERM U114, F-75231 Paris, France (M.M.D.);Department of Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania 19104 (J.-L.M.); Departement de Psychiatrie, Universite de Geneve, CH-1225 Geneve (Y.C., C.B.); Preclinical Research, Sandoz Pharma Ltd, CH-4002 Base1 (J.M.P., M.M.D.);and Institut de Physiologie, Universite de Lausanne, CH-1005 Lausanne (P.J.M., J.-L.M.), Switzerland

ABSTRACT The distribution of vasoactive intestinal peptide (VIP) binding sites in the pigeon brain was examined by in vitro autoradiography on slide-mounted sections. A fully characterized monoiodinated form of VIP, which maintains the biological activity of the native peptide, was used throughout this study. The highest densities of binding sites were observed in the hyperstriatum dorsale, archistriatum, auditory field L of neostriatum, area corticoidea dorsolateralis and temporo-parieto-occipitalis, area parahippocampalis, tectum opticum, nucleus dorsomedialis anterior thalami, and in the periventricular area of the hypothalamus. Lower densities of specific binding occurred in the neostriatum, hyperstriatum ventrale and nucleus septi lateralis, dorsolateral area of the thalamus, and lateral and posteromedial hypothalamus. Very low to background levels of VIP binding were detected in the ectostriatum, paleostriatum primitivum, paleostriatum augmentaturn, lobus parolfactorius, nucleus accumbens, most of the brainstem, and the cerebellum. The distribution of VIP-containing fibers and terminals was examined by indirect immunofluorescence using a polyclonal antibody against porcine VIP. Fibers and terminals were observed in the area corticoidea dorsolateralis, area parahippocampalis, hippocampus, hyperstriatum accessorium, hyperstriatum dorsale, archistriatum, tuberculum olfactorium, nuclei dorsolateralis and dorsomedialis of the thalamus, and throughout the hypothalamus and the median eminence. Long projecting fibers were visualized in the tractus septohippocampalis. In the brainstem VIP immunoreactive fibers and terminals were observed mainly in the substantia grisea centralis, fasciculus longitudinalis medialis, lemniscus lateralis, and in the area surrounding the nuclei of the 7th, 9th, and 10th cranial nerves. The correlation between the distribution of VIP binding sites and immunoreactive fibers and terminals was assessed in a restricted number of regions. A qualitatively good matching was found in the area corticoidea dorsolateralis, hyperstriatum dorsale, hyperstriatum accessorium, nucleus septi lateralis, nuclei dorsomedialis and dorsolateralis thalami, and in some hypothalamic areas. A striking mismatch occurred in the hyperstriatum ventrale, neostriatum, tectum opticum (high to moderate density of binding sites but only few immunoreactive profiles), and in the tuberculum olfactorium, median eminence, and spinal cord (lower density of binding sites but abundant immunoreactive profiles). The paleostriatum, lobus parolfactorius, and ectostriatum were virtually devoid of both binding sites and immunoreactive profiles. The results are discussed in

Accepted November 9, 1990. Address reprint requests to Patrick R. Hof, Fishberg Research Center for Neurobiology, Box 1065, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York,NY 10029.

o 1991 WILEY-LISS, INC.

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relation to the known actions of VIP in the rodent and avian brain and are compared with previous observations on the distribution of VIP binding sites in the central nervous system of other vertebrates. Key words: VIP, autoradiography, immunohistochemistry, neuropeptides, avian brain

Vasoactive intestinal peptide (VIP) is a 28-residue peptide originally isolated from porcine duodenum (Said and Mutt, '70). The presence ofVIP has also been demonstrated in the respiratory, genitourinary, and endocrine systems (Larsson et al., '77, '78; Ahren et al., '80; Costa et d . , '80; Knight et al., '87). VIP has also been detected in the central nervous system ( CNS) where it is predominantly contained in locally projecting neurons (Fuxe et al., '77; Lor6n et al., '79; Sims et al., '80; Morrison et al., '84; Abrams et al., '85; Magistretti and Morrison, '85, '88; Gall et al., '86). Some long VIP-immunoreactive projections have also been described in the rat CNS (Roberts et al., '80; Marley et al., '81; Eiden et al., '85; Nahin et al., '88). Furthermore, specific binding sites for VIP have been demonstrated in synaptosoma1 membranes from various areas of the rodent brain (Robberecht et al., '78; Taylor and Pert, '79; Staun-Olsen et al., '82, '85; Ogawa et al., '85; Roth and Beinfeld, '85) and by in vitro autoradiography in the brain of various vertebrate species (Besson et al., '86; Martin et al., '87; Magistretti et al., '88; Diet1 et al., '90). In the rat brain, VIP receptors are predominantly associated with areas involved in the processing of specific sensory inputs; furthermore, a good matching exists in numerous CNS areas between VIP immunoreactive profiles and binding sites (Martin et al., '87). Cellular VIP-mediated actions have been reported, particularly in the cerebral cortex. Thus in the rodent neocortex, VIP stimulates cyclic adenosine-monophosphate (CAMP) synthesis and promotes glycogen hydrolysis (DeschodtLankmann et al., '77; Magistretti et al., '81; Magistretti and

Schorderet, '84, '85; Magistretti, '86). VIP interacts synergistically with noradrenaline to stimulate CAMPformation and to depress firing of cortical neurons (Magistretti and Schorderet, '84, '85; Ferron et al., '85; Magktretti and Morrison, '88). VIP also enhances glucose utilization in the rat striatum and cingulate cortex (McCulloch et al., '83; McCulloch and Kelly, '83). Metabolic effects of VIP have been reported on cultured astrocytes and brain microvessels preparations (McCulloch and Edvinsson, '80; Wei et al., '80; Magistretti et al., '83; Huang and Rorstad, '83, '84). VIP has been shown to affect neurotransmitter release in the rodent hypothalamus (Kato et al., '78; Besson et al., '79; Epelbaum et al.,'79; Enjalbert et al., '801, as well as in the avian hypothalamus (Proudman and Opel, '83; Hall and Chadwick, '85; Macnamee et al., '86). VIP also displays a vasodilatory action in the isolated gill of the trout (Bolis et al., '84a,b). The sequence of VIP is highly preserved across species (Dimaline and Dockray, '82). However, a variant of VIP has been isolated from the chicken intestine (Nilsson, '74). This VIP species differs from porcine VIP by five amino acids (Nilsson, '75). VIP has recently been shown by immunohistochemistry to occur in certain areas of the avian central and peripheral nervous system (Karten et al., '82; Yamada et al., '82; Ball et al., '86, '88; Macnamee et al., '86; Wingfield et al., '86; Reiner, '87; Du et al., '88; Silver et al., '88; Walcott et al., '89; Shimizu and Karten, '90). In the present study, we have examined the distribution of VIP binding sites by in vitro autoradiography and the localization of VIP-containing fibers and terminals by immunohistochemistry in the pigeon brain in an attempt to

Abbreviations Acb AD

AHP Ai Aid AP

APH

AV bcd Cb CDL CO da DLL DMA E EM fa FRL GCt HA HD HM HP

Hv

INP

I0 IPC

nucleus accumbens archistriatum dorsale area hypothalami posterioris archistriatum intermedium archistriatum intermedium pars dorsalis archistriatum posterius area parahippocampalis archistriatum ventrale brachium conjonctivum descendens cerebellum area corticoidea dorsolateralis cbiasma opticum tractus archistriatalis dorsalis nucleus dorsolateralis anterior thalami, pars lateralis nucleus dorsomedialis anterior thalami ectostriatum median eminence tractus frontoarchistriatalis forniatio reticularis lateralis mesencephali substantia grisea centralis hyperstriaturn accessorium hyperstriatum dorsale nucleus habenularis medialis hippocampus hyperstriatum ventrale nucleus intrapeduncularis nucleus isthmoopticus nucleus istbmi, pars parvicellularis

L LC LHY LPO N NC NI

nx

PA PMH PP qf R Rt S

SAC SGF SGP SL SLU SOP TeO Tn TO

TrO VIP VIP-ir

rose's auditory field L nucleus locus coeruleus nucleus lateralis hypothalami lobus parolfactorius neostriatum neostriatum caudale neostriatum intermedium nucleus nervi X paleostriatum augmentatum nucleus medialis hypothalami posterioris paleostriatum primitivurn tractus quintofrontalis nucleus raphes nucleus rotundus nucleus tracti solitarii stratum album centrale stratum griseum et fibrosum superficiale substantia grisea et fibrosa periventricularis nucleus septi lateralis nucleus semilunaris stratum opticum tectum opticurn nucleus taeniae tuberculum olfactorium tractus opticus vasoactive intestinal peptide vasoactive intestinal peptide-immunoreactive

VIP IN THE PIGEON BRAIN

Fig. 1. Distribution of specific VIP binding sites in the pigeon brain. A. Coronal section at the level of the nucleus septi lateralis. B. Coronal section at the level of the auditory field L. C. Coronal section through the tectum opticum. D. Horizontal section at the level of the nucleus dorsomedialis anterior thalami. Colors show differences in VIP binding

395

sites densities. A semiquantitative assessment of the densities is given by the optical density scale. Each section is a picture of a separate computer screen display. Extremes of scale: blue = lowest density, white = highest density.

P.R. HOF ET AL.

396

HA

\

Acb

TO SL

A 10.00 Fig. 2. Schematic comparisons of VIP binding sites (left side) and VIP-ir fibers and terminals (right side) distribution in the pigeon brain. Coronal section at the level of the nucleus septi lateralis. The anteriopos-

terior coordinates in the lower left corner correspond to a plate from the atlas of Karten and Hodos ('67). Labeling patterns are shown on Figure 5.

correlate the pre- and postsynaptic markers for VIP circuits within the CNS of an avian species.

(M-'"I-VIP), with a high specific activity (2050 Ci/mmol) and maintaining the full biological activity of the native peptide (Martinet al., '86).

MATERIALS AND METHODS Animals

Autoradiographic procedure

Sixteen adult male pigeons (Columba livia, 400-500 g, 4 years old), were used throughout this study. They were purchased from S. Abdel'Al, Basel, Switzerland, and from F. Carra, Rontalon, France.

Ligand preparation VIP was labeled with sodium ['251]iodideusing the chloramine-T method and purified by reverse phase highperformance liquid chromatography (RP-HPLC) according to the method ofMartin et al., ('86). Briefly, the labeled VIP mixture was passed through a Sep-pack C,, cartridge and further purified on a RP-HPLC system (Waters, UK). The column used was a Radialpak p-Bondapak C,, cartridge and the solvents were 0.1% TFA in CH,CN chromatography grade. The equilibrated column was first eluted isocratically with 74% of 0.1% TFA and 26% CH,CN. After 15 minutes, a linear gradient of CH,CN was started, 26-32% over 1 hour. This method allows for the preparation of a labeled VIP form monoiodinated on Tyr'', oxidized on Met'7

The autoradiographic procedure has been previously described in detail (Wamsley and Palacios, '83). Briefly, nine pigeons were killed by decapitation, their brains rapidly removed and frozen on dry ice. Ten-pm-thick sections were cut on a Leitz 1720 microtome-cryostat, mounted onto gelatin-coated microscope slides, and stored at -20°C until used. Slide-mounted brain sections were then brought to room temperature and incubated for 20 minutes in the presence of 60 pM of M-1251-VIPin 50 mM Tris-HC1 buffer pH 7.4 containing 5 mM MgCl,, 2 mM EGTA, 0.1 mM bacitracin, and 0.2% bovine serum albumin (RIA grade). Nonspecific binding was determined in the presence of 1 p,M unlabeled VIP. After incubation, the slides were rapidly dipped into cold deionized water, rinsed in ice-cold incubation buffer for two 4-minute baths, dipped again in cold water, and finally dried under a stream of cold dry air. The dried slidemounted sections were then apposed to [3H]-sensitiveUltrofilms as previously described (Wamsley and Palacios, '83) and exposed for 60 hours at 4°C before development. Initial

VIP IN THE PIGEON BRAIN

397

APH

A 6.25 Fig. 3. Schematic comparisons of VIP binding sites (left side) and VIP-ir fibers and terminals (right side) distribution in the pigeon brain. Coronal section through the nucleus rotundus. The anterioposterior coordinates in the lower left corner correspond to a plate from the atlas of Karten and Hodos ('67). Labeling patterns are shown on Figure 5

kinetics and pharmacological experiments (Martin et al., '87) have shown that the binding of M-lZ51-VIPto rat brain slide-mounted sections was time-dependent, saturable, and reversible. Association of M-lZ5I-VIPspecific binding was maximal within 90 to 120 minutes. The saturation of specific binding corresponded to approximately 50% of total binding and had a high affinity (K, of 76.6 pM) and a low capacity (in the fmol/mg protein range). Dissociation of M-'251-VIPwas maximal after 10 minutes. Unlabeled VIP and the structurally related peptides PHI and secretin competed in a concentration-dependent manner for binding sites labeled by M-lZ5I-VIPwith the following rank order of potencies: VIP > PHI > secretin. Finally, M - T - V I P was shown by RP-HPLC analysis to be stable after 90 minutes of incubation in the incubation buffer (Martin et al., '87). Autoradiographic experiments carried out in parallel in

different vertebrate species including bird revealed that association kinetics, percentage of specific binding, and competition with related peptides of M-1251-VIPon slidemounted brain sections were comparable in all the species investigated (Magistretti et al., '88; Diet1 et al., '90). The density of VIP binding sites in the different structures of the pigeon brain was semiquantitatively assessed using a neuroimaging system consisting of a Zeiss AxioPlan photomicroscope and CCD Television Camera (Tokina CP3000) connected to a Compaq Deskpro 386/20 microcomputer and a SAMBAm image analysis system (Brugal, '84) developed by TITN Inc. (Grenoble, France).

Immunohistochemistry Seven pigeons were used for immunohistochemical analysis. Each animal was deeply anesthetized with sodium

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HA

APH

D

i DMA

J APH

A 5.50

DLL

EM

Fig. 4. Schematic comparisons of VIP binding sites (left side) and VIP-ir fibers and terminals (right side) distribution in the pigeon brain. Coronal section at the level of the median eminence. The anterioposte-

rior coordinates in the lower left corner correspond to a plate from the atlas of Karten and Hodos ('67). Labeling patterns are shown on Figure 5.

pentobarbital (30 mgkg intraperitoneally) and perfused transcardially with 100 ml of Tyrode's solution followed immediately by 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.4 (PB) at 20°C. The brain was removed, postfixed for 2 hours in the same fixative and kept overnight in cold PB containing 10% sucrose. Serial 10 pm-thick coronal sections were cut on a ReichertJung microtome-cryostat, collected on gelatin-coated microscope slides, and processed for indirect immunofluorescence as previously described (Coons, '58; Du et al., '87, '88). Briefly, sections were incubated for 1 hour a t 20°C in the presence of 1:1,000 rabbit VIP antiserum diluted in PB containing 0.3%Triton X-100. After several washes in PB, the sections were incubated for 1 hour with FITCconjugated antirabbit IgG, diluted in PB a t a working dilution of 1:200. The preparation and characterization of the VIP antiserum have been extensively reported elsewhere (Chayvialle et al., '83; Charnay et al., '85). This

antiserum preferentially binds the C-terminal portion of VIP and does not cross-react with several other neuropeptides, including structurally related peptides such as PHI, glucagon, and secretin (Chayvialle et al., '83; Charnay et al., '85). The specificity of the immunoreaction was routinely assessed by omitting the first step of the immunoreaction or by preabsorption of the primary antiserum with natural porcine VIP (0.1-1 pM).Identification of VIP in the avian CNS with the used antibody has been previously documented (Du et al., '88). Some sections were stained with cresyl violet for identification of the anatomical structures. Nomenclature was adapted from Karten and Hodos, ('67). The line drawings on Figures 2-5 were generated from a computerized atlas of the pigeon brain adapted from that of Karten and Hodos, ('67). This atlas was prepared with the aid of a software facility developed at the Department of Neuropharmacology, Research Institute of Scripps Clinic, La Jolla, California, and subsequently edited with a commer-

VIP IN THE PIGEON BRAIN

399

TABLE 1. Semiquantitative Analysis of M - 1 2 5 1 - ~Binding P Site Densities in the Pigeon Brain' Regions Area corticoideadorsolateralis Hyperstriatum accessorium Hyperstriatum dorsale Hyperstriaturn ventrale Neostriatum (field L) Neostriatum intermedium Nwstriatum caudale Archistriatum dorsale Archistriatum ventrale Archistriatum intermedium pars dorsalis Archistriatum posterius Archistriatum mediale Nucleus taeniae Ectostriatum Paleostriatum augmentatum Paleostriatum primitivum Lobus parolfactorius Nucleus basalis Nucleus septi lateralis Nucleus accumbens Tuberculum olfactorium Nucleus preopticus medialis Area posteromedialis hypothalami Area periventricularishypothalami Nucleus dorsomedialisanterior thalami Nucleus dorsolateralisanterior thalami Stratum griseum et fibrosum superficiale Stratum album centrale Nucleus semilunaris Nucleus raphes Nucleus locus coeruleus Cerebellum (cortex) Lamina frontalis superior Chiasma opticum Nonspecific binding

%T 69.3 5 3.7 42.5 t 1.9 59.7 5 4.1 70.8 t- 5.7 38.0 5 2.5 65.8 t 1.9 75.3 2 3.0 47.6 f 6.2 67.4 5.2 30.8 5 1.7 30.1 f 1.4 67.3 f 3.8 34.2 f 2.5 94.0 2 4.9 95.9 2 3.0 99.1 2 0.8 95.1 ? 5.3 58.5 f 2.3 68.2 f 5.1 92.9 f 5.6 64.0 f 1.7 82.0 f 2.5 89.4 f 5.6 53.0 ? 1.8 39.9 t 6.0 52.5 -t 4.2 7.3 f 0.2 84.5 f 3.4 50.2 f 0.5 81.1 2 4.4 91.7 ? 0.2 92.5 f 7.2 99.3 f 0.5 98.1 ? 1.2 97.8 ? 0.9

*

'Results are expressed as means ? SEM from 9 animals. Dependmg on the area, series of 4 to 14 measurements were performed. Values represent the percentage of light transmittance through a given structure (BT: a high value means a low optical density and a low grain density) and were calculated from an arbitrary gray scale (0 = white to 255 = black) using a computer-assisted image analysis system.

cially available graphic illustration software (ADOBE Illustrator 8aTM) on a Macintosh I1 microcomputer. Porcine VIP was purchased from Professor V. Mutt, Karolinska Institutet, Stockholm,Sweden. Sodium ['251]iodide(specificactivity 13-16.9 mCi/pg iodine) was obtained from Amersham International plc, and Ultrofilms from LKB (Sweden). The FITC-conjugated antirabbit IgG was from Miles Ltd. All other substances were from commercial sources.

temporo-parieto-occipitalis were also enriched in VIP binding sites (Figs. lA,B,D, 3,4),as was the nucleus basalis. The nucleus septi lateralis presented relatively high densities of M-lZ5I-VIPbinding sites, whereas very low labeling was detected in the nucleus accumbens (Figs. lA, 2). A stripe of very high grain densities was observed on the lateral aspect of the ventricular wall. In the thalamus, the highest densities of M-lZ5I-VIPbinding sites were found in the nucleus habenularis medialis, and nucleus dorsomedialis anterior thalami, whereas lower densities were observed in the nucleus dorsolateralis anterior thalami (Figs. lB,D, 3, 4). The nucleus rotundus was devoid of specificVIP recognition sites. In the hypothalamus, a band of intense labeling was revealed in the periventricular area (Figs. lB, 3 , 4 ) ,and moderate to low densities of binding were observed in the nucleus posteromedialis hypothalami and in the lateral hypothalamic area (Figs. 3 , 4 ) . The tectum opticum presented the highest densities of VIP binding sites in the whole pigeon brain (Figs. 1C,D, 4, 5). They were concentrated in the stratum griseum et fibrosum superficiale and in the stratum griseum centrale, whereas the stratum album centrale was only moderately labeled and the stratum griseum profundum was devoid of VIP binding sites (Figs. lC,D, 4, 5). In the remaining brainstem, only a few structures were found to contain VIP specific binding sites. The highest binding levels were observed in the nucleus semilunaris (Figs. lC, 5). Low grain densities were visualized in the nucleus locus coeruleus, nucleus raphes, and nucleus isthmoopticus (Figs. lC, 5). The cerebellum contained very low densities of specific binding sites (Figs. lC, 5). No specific binding was found in white matter tracts such as tractus frontoarchistriatalis, tractus quintofrontalis, and in the cell-free laminae that separate the major avian telencephalic divisions (Figs. 1,2). Figure 6A,B illustrates the total and nonspecific binding levels, determined in the presence of 1FM unlabeled VIP in the incubation medium on two adjacent sagittal sections through the pigeon brain.

VIP immunohistochemistry

A high density of VIP-immunoreactive (VIP-ir)fibers and terminals was found in the hyperstriatum accessorium, hyperstriatum dorsale, hippocampus, and archistriatum (Figs. 2-4, 7B, 8). Few VIP-ir fibers were visualized in the hyperstriatum ventrale (Fig. 8). VIP-ir fibers were also RESULTS observed in the area parahippocampalis and in the area Autoradiographic distribution of corticoidea dorsolateralis (Figs. 3, 4, 71, where fine and VIP binding sites varicose fibers were found to form basketlike structures In the telencephalon, the highest grain densities were probably around cell bodies (Fig. 7A, arrows). VIP-ir fibers visualized in the hyperstriatum accessorium, hyperstria- were more numerous in the archistriatum ventrale than in tum dorsale and ventrale as well as throughout the neostri- the archistriatum dorsale. The tuberculum olfactorium was atum (Figs lA,B,D, 2-4; see also Fig. 6A).In the archistria- enriched in very thin VIP-ir fibers and terminals (Fig. 2). tum dorsale and ventrale, very high levels of specific The area septalis lateralis was enriched in long and thin binding sites were also detected (Figs. lB, 3, 4). The varicose fibers, which seemed to project through the tractus archistriatum intermedium pars dorsalis, archistriatum septohippocampalis. Similar immunoreactive fibers were posterius, and nucleus taeniae had a comparable density of also observed coursing toward the tuberculum olfactorium. binding sites (see Fig. 6A). The archistriatum mediale was High densities of VIP-ir fibers were present in the nucleus characterized by relatively lower grain densities (Table 1; accumbens and along the ventricular wall of the lobus see Figs. lB, 6A). The ectostriatum displayed low densities parolfactorius (Fig. 2). In the nuclei dorsomedialis anterior of binding sites (Figs. IA, 2). The paleostriatum augmenta- and dorsolateralis anterior thalami, fine and widespread tum and lobus parolfactorius contained very low densities VIP-containing fibers and terminals were observed (Figs. 4, and the paleostriatum primitivum was practically devoid of 9).VIP-positivefibers were observed in the nucleus preoptiVIP recognition sites (Figs. lA, 2). The nucleus intrapedun- cus anterior, nucleus anteromedialis, nucleus supraopticus, cularis was strongly labeled (Figs. lA, 2 ) . The area parahip- nuclei posteromedialisand posterolateralis, nucleus perivenpocampalis and the area corticoidea dorsolateralis and tricularis pars magnocellularis, and area lateralis hypothal-

P.R. HOF ET AI,.

400

SAC '

R

SLU

A 2.25

Binding sites Very high density

Fibers

@

High density

[3

Moderatedensity

0 Very low density 0 Fig. 5. Schematic comparisons of VIP binding sites (left side) and VIP-ir fibers and terminals (right side) distribution in the pigeon brain. Coronal section through the tectum opticum. The anterioposterior coordinates in the lower left corner correspond to a plate from the atlas of Karten and Hodos ('67).

VIP IN THE PIGEON BRAIN

401

Fig. 6. Coronal adjacent sections through the pigeon brain at the level of the auditory field L. A. Total binding of M-’*’I-VIP. Subdivisions of the archistriaturn are labeled according to the nomenclature of Zeier and Karten (’71). B. Nonspecificbinding observed in the presence of 1pM unlabeled VIP in the incubation medium.

ami (Figs.3,4,10,11). The nuclei tuberis and infundibularis were enriched in fine VIP-positive fibers, close to the lateral border of the ventricle. Finally, the median eminence displayed a lining of thick VIP-ir fibers on its external aspect (Figs. 4, 11). The tectum opticum contained very few, scattered immunoreactive fibers confined to the stratum griseum et fibrosum superficiale; the cerebellum was completely devoid of VIP-ir elements (Figs. 4, 5). In the brainstem, some immunoreactive fibers were observed in the substantia grisea centralis and in the tegmentum dorsale (Fig. 5). There were scattered fibers in the formatio reticularis lateralis, lemniscus lateralis, fasciculus longitu-

dinalis medialis, raphe area, nucleus subcoeruleus, whereas the nuclei of the 7th cranial nerve, nucleus vestibularis medialis, and sensory nuclei of the gth and loth cranial nerves were characterized by a high density of VIPcontaining fibers (Figs. 5, 12). Intensely labeled VIP-ir cell bodies were observed along the rostra1 pole of the lateral ventricle, in the nucleus septi lateralis (Fig. 131, medial and anterior hypothalamic areas, and median eminence. A few less intensely labeled cells were found in the region of the locus coeruleus and area postrema. It should be noted that the absence of colchicine treatment may explain the relative paucity of VIP-ir cell bodies observed in our materials.

fim.

Fig. 7. A. VIP-ir profiles in area corticoidea dorsolateralis. Note the fine varicose basketlike elements (white arrows). B. VIP-ir fibers in the archistriatum ventrale. The black arrows on the inset show the location of fields A and B. Bar = 110

Fig. 8. Photomontage through the hyperstriaturn accessorium, hyperstriatum dorsale, and hippocampus. Note the numerous labeled fibers in these areas and the occurrence of inimunoreactive fibers in the vicinity of the ventricle border. The inset shows the approximate location of the photomontage. Bar = 110 wm.

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Fig. 9. VIP-ir fibers in the thalamus. Note the very high density of fibers in the nucleus dorsomedialis anterior. The nucleus medialis hypothalami posterioris contains also a large number of VIP-ir elements. The inset shows the approximate location of the photomontage. Bar = 110 km.

DISCUSSION In the present study we have examined the distribution of VIP recognition sites by in vitro autoradiography in the pigeon brain, in parallel with the localization of VIP-ir fibers and terminals. For this purpose we used a fully characterized RP-HPLC purified monoiodinated derivative form of VIP (Martin et al., '86, '87) and a previously described polyclonal antibody against VIP (Chayvialleet al., '83; Charnay et al., '85). Previous results have indicated that VIP binding sites appear quite early in the phylogeny and that they are already present in the snake and frog, but not in the teleost,

brain (Magistretti et al., '88; Diet1 et al., '90). VIP-ir structures have been observed in several structures of the avian CNS. In the chick, VIP-ir neurons and fibers have been described in the spinal cord in lamina I of the dorsal horn and in the lateral funiculus (Du et al., '88), in the medulla at the level of the nucleus of the lothcranial nerve (Ambrosiet al., '841, and more rostrally in the brainstem, in the substantia grisea centralis and in the tegmentum (Nicolardi et al., '84). In the hen, quail, duck, and pigeon hypothalamus, VIP-ir elements have been reported in the nucleus infundibularis, median eminence, nucleus preopticus, nucleus supraopticus, nucleus paraventricularis, and

VIP IN THE PIGEON BRAIN

Fig. 10. VIP-ir profiles in the lateral and posterior hypothalamic areas. Note the presence of a few labeled cell bodies. The insert shows the approximate location of the photomontage. Bar = 110 Fm.

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Fig. 11. VIP-ir profiles in the median eminence. There is a dense labeling on the external aspect of the median eminence. Note the presence of scattered VIP-ir cell bodies. Bar = 110 pm.

in the nucleus mammillaris lateralis (Yamada et al., '82; Korf and Fahrenkrug, '84; Mikami, '86; Macnamee et al., '86; Silver et a]., '88). Some positive neurons and fibers have been observed in the nucleus septi lateralis of the quail (Yamada et al., '821, duck (Korf and Fahrenkrug, '841, and ring dove (Silver et al., '88). In the present study we have also observed VIP-ir cell bodies in these regions. VIP-ir profiles have been observed in the deep layers of the tectum and in the substantia grisea centralis (Karten et al., '82). Furthermore, VIP-ir neurons in the nucleus of EdingerWestphal innervating the ciliary ganglion (Reiner, '87), and fibers innervating various ocular structures including blood vessels have been described (Reiner, '87; Walcott et al., '89).

In addition, VIP-ir profiles have been visualized in different areas involved in vocal control in songbirds, particularly in the nucleus caudalis of the ventral hyperstriatum, the nucleus magnocellularisof the anterior neostriatum, around the nucleus robustus in the archistriatum, and in the nucleus intercollicularis in the mesencephalon (Ball et al., '88). The correlation between the distribution of VIP recognition sites and VIP-ir profiles was analyzed. Figures 2-5 can be used for direct comparison of the density patterns. The regions where the most striking qualitative matching was observed were the periventricular areas, the nucleus septi lateralis, and the nucleus dorsomedialisthalami.

VIP IN THE PIGEON BRAIN

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Fig. 12. VIP-ir profiles at the level of the dorsal motor nucleus of the lothnerve. Note the dense plexus of VIP-ir fibers in the sensory division of the lothnerve. Bar = 110 um.

The periventricular zone in the hypothalamus displayed a high grain density and numerous thin fibers and terminals. The nucleus septi lateralis was characterized by the presence of a small area highly enriched in VIP binding sites, along the wall of the ventricle. In the same area numerous VIP-ir fibers were visualized. Previous reports have demonstrated the presence of VIP immunoreactivity in fibers and neurons in these periventricular areas in several avian species (Yamada et al., '82; Korf and Fahrenkrug, '84; Macnamee et al., '86; Silver et al., '88). In the hypothalamus, intermediate densities of VIP binding sites were present in the lateral hypothalamic area, whereas scattered VIP-positivefibers were observed throughout the hypothalamus. The nuclei dorsomedialis anterior and dorsolateralis anterior thalami revealed high densities of specific binding. In these nuclei, numerous VIP-ir fibers were detected. The area corticoidea dorsolateralis presented both high levels of VIP binding sites and VIP-ir fibers and terminals. A strong matching was observed in our materials in parts of the archistriatum, hyperstriatum accessorium, and hyperstriatum dorsale. The remaining hyperstriatum and neostriatum presented high densities of VIP receptors but no detectable or few VIP-ir structures. Comparable mismatches were found in the external layers of tectum opticum, brainstem, and spinal cord. Mismatches between the presence of specific receptors and the neurotransmitter is frequently observed in the CNS (Herkenham and McLean, '86; Herkenham, '88). However, an unusually pronounced matching for VIP-ir profiles and recognition sites has been reported in the rat brain (Martin et al., '87). In this species, the codistribution of VIP immunoreactivity and binding sites was observed in the olfactory bulb, throughout the neocortex, in the hippocampus, amygdala, lateral septum, nucleus accumbens, some thalamic and hypothalamic nu-

clei, habenula, superior colliculus, locus coeruleus, and area postrema (Martin et al., '87). Furthermore, a noticeable overlap in the distribution of opiate receptors and opiatecontaining fibers has been described in the striatal portion of the basal ganglia in the pigeon (Reiner et al., '89). In addition, in the nucleus lateralis spiriformis of the same species, a considerable matching has been observed for GABA and neurotensin markers (Brauth et al., '86). The mismatch between VIP-ir structures and binding sites that is observed in some regions of the pigeon brain may be related to the fact that colchicine was not used in the present studies and hence that VIP-ir cell bodies or fibers may not have been visualized. It is worth noting that topographically comparable structures presented similar autoradiographic labeling patterns in rodent and avian brain. Thus although in birds and reptiles the pallium does not develop following the same anatomical lines as the mammalian neocortex (Kiilkn, '53; Karten, '69; Nauta and Karten, '70; Northcutt, '81; Reiner et al., '84), functional similarities between mammalian neocortex and parts of the avian dorsal ventricular ridge and Wulst can be considered (Karten and Shimizu, '89). For instance, the rodent cerebral neocortex displayed an overall fairly high density of VIP recognition sites (Martin et al., '87; Magistretti et al., '88), and in the pigeon brain, the area corticoidea dorsolateralis and the area temporo-parietooccipitalis were enriched in VIP binding sites. Some functionally specific areas, such as visual Wulst and Rose's auditory field L showed high densities of VIP receptors, whereas the ectostriatum, considered to be the homologue of layer IV of the mammalian extrastriate visual area 18 (Nauta and Karten, '70; Brauth et al., '861, displayed only a low level of binding sites. For example, in the mammalian brain, the primary and secondary visual, primary auditory

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408 -~

Fig. 13. VIP-ir cell bodies along the ventricular wall in the nucleus septi lateralis. Sagittal section. Bar = 110 km. The inset shows a higher magnification of the cell bodies. Arrows identify the same cell. Bar (inset) = 20 km.

as well as motor and somatosensory neocortical areas were all enriched in VIP binding sites (Martin et al., '87, Diet1 et al., '90). The major motor somatic region of the pigeon brain (dorsolateral archistriatum) contains high levels of VIP receptors. The archistriatum ventrale has been related to the mammalian amygdaloid complex (Nauta and Karten, '70; Zeier and Karten, '71). The rodent amygdala and pigeon archistriatum ventrale were both densely labeled. The periventricular areas and lateral septum displayed the same levels of grains densities in rodent and avian brain.

The paleostriatal complex is the counterpart of the mammalian basal ganglia (Nauta and Karten, '70; Reiner et al., '84). Here, too, similarities of labeling were observed: the rodent caudate-putamen contains higher VIP binding sites densities than globus pallidus (Martin et al., '87). In the pigeon brain, the lobus parolfactorius and paleostriat um augmentatum were slightly more enriched in VIP receptors than the paleostriatum primitivum where binding sites were virtually undetectable. It should be noted, however, that the VIP binding sites densities in these areas were

VIP IN THE PIGEON BRAIN much lower in the pigeon brai6 as compared to the rodent brain. Very high densities of labeling were observed in the rodent superior colliculus and in the avian tectum opticum. These similarities of VIP receptors distribution can be extended to some degree to other mammals such as the cat and monkey (Dietl et al., '90). A comparable interspecies conservation of the distribution of dopamine receptors has been reported in the basal ganglia (Richfield et al., '87; Dietl and Palacios, '88a,b). Similar observations have been made for muscarinic and GABA/benzodiazepine receptors (Dietl et al., '88a,b). We have previously reported that VIP receptors in the rodent CNS are associated with primary sensory areas of the neocortex and to different regions involved in the processing of specific sensory information such as the olfactory bulb, lateral and medial geniculate nuclei, lateral and medial ventroposterior thalamic nuclei, habenula, superior colliculus, cochlear and vestibular nuclei, and nucleus of the solitary tract (Martin et al., '87; Magistretti et al., '88; Dietl et al., '90). It is worth noting that in the pigeon brain some specific sensory areas contain high densities of VIP binding sites. These areas include part of the primary auditory field L, the visual areas within the Wulst, somatosensory areas in the neostriatum intermedium and caudale, the retinorecipient layers of tectum opticum, the retinorecipient nucleus dorsolateralis anterior thalami (Nauta and Karten, '701, and the nucleus basalis, which receive sensory information of the beak via a direct projection from the nucleus principalis nervi trigemini (Wild, '87). Several cellular actions of VIP have been described in the avian CNS. VIP has been reported to be a physiological prolactin-releasing factor (Macnamee et al., '86). A similar action was previously demonstrated in the rat hypothalamus (Kato et al., '78). Several neuroendocrine effects of VIP have been described in the rodent hypothalamus. Thus VIP inhibits the release of somatostatin (Epelbaum et al., '79) and stimulates LHRH release from median eminence synaptosomes (Besson et al., '79). In the avian brain, VIP may also interact with the release of LHRH, TRH, and somatostatin into the portal system, in view of the dense distribution of VIP-immunoreactive profiles in the median eminence and the presence of VIP-positive terminals on the wall of portal vessels (Yamada et al., '82). VIP may also contribute to the blood flow regulation in the area of the nucleus infundibularis (Yamada et al., '82). VIP-ir profiles and VIP-binding sites have been demonstrated in close contact with the ventricular wall in the rodent as well as in the avian brain (Kohler, '83; Korf and Fahrenkrug, '84; Martin et al., '87; Silver et al., '88). The ependymal and neuronal specializations of the ventricular aspect of the avian area septalis have been considered as a circumventricular organ (lateral septa1 organ of Kuenzel and van Tienhoven ('82); see also Korf and Fahrenkrug, ('84)). In our study this particular area showed very high densities of VIP specific binding sites and VIP-ir material. It is interesting to note that in the rodent, the circumventricular organs were heavily labeled (Martinet al., '87) and that CSF-contacting VIP-ir fibers have been described in the rat gyms dentatus (Kohler, '83). The physiological significance of these VIP-ir CSF-contacting fibers is unknown. Recently, however, VIP has been shown to be colocalized with opsinlike immunoreactive material in some neurons and fibers in the area septalis, close to the ventricular wall and in the infundibulum of different bird species (Silver et al.,

409 '88). The authors suggest that these cells might be involved in extraocular photoreception, since in several avian species induction of gonadal growth and regression has been shown to be controlled by encephalic photoreceptors {for review, see Groos ('SZ)}. Finally, VIP has been localized in areas involved in vocal control in two wild songbird species (Ball et al., '88). In these species, VIP was visualized in particular in sexually dimorphic telencephalic areas located in the hyperstriatum ventrale, archistriatum, and anterior neostriatum. It was also present in the nucleus intercollicularis, which is implicated in the motor control of song (Ball et al., '88).Although our study did not reveal VIP immunoreactivity in these areas, results reported by Ball et al., ('88) provide some evidence for a role of VIP in the production of song at least in some passerine species. Seasonnal variations in neuropeptides content have been previously described in the CNS of some avian species (Ball et al., '88). The present set of data provides a detailed mapping of VIP recognition sites and of VIP-ir fibers and terminals in the pigeon brain. These results could be of value for future investigations on the comparative aspects of the cellular function and regional specializations of VIP-containing systems in the vertebrate brain.

ACKNOWLEDGMENTS The authors thank R. Guntern, I. Mikolajewski, M. Surini, B. Greggio, P.Y. Vallon, M. Rigo, and F. Pillonel for expert technical assistance; Drs. F.E. Bloom and W.G. Young for help during the design of the computerized atlas of the pigeon brain; and P. Leroux for skillful assistance with the graphic illustrator. This research was supported by a grant from Fonds National Suisse de la Recherche Scientifique (FNRS) No 3.357-0.86 to P.J.M. P.R.H. and J.L.M. were the recipients of FNRS fellowships.

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