Atrial natriuretic peptide in the central nervous system of the rat

Cellular and Molecular Neurobiology, Vol. 8, No. 4, 1988

Review and Commentary

Atrial Natriuretic Peptide in the Central Nervous System of the Rat

G e r h a r d Skof i t s ch I and D a v i d M . J a c o b o w i t z 2

Received February 2, 1988, accepted February 10, 1988

KEY WORDS: atrial natriuretic peptide; central nervous system; immunoeytochemistry; HPLC; radioimmunoassay; autoradiography; intraeerebral injection.

S U M M A R Y

1. Studies of the presence of atrial natriuretic peptide immunoreactivity and receptor binding sites in the central nervous system have revealed unusual sites of interest.

2. As a result, numerous studies have appeared that indicate that brain atrial natriuretic peptide is implicated in the regulation of blood pressure, fluid and sodium balance, cerebral blood flow, brain microcirculation, blood-brain barrier function, and cerebrospinal fluid production.

3. Alteration of the atrial natriuretic peptide system in the brain could have important implications in hypertensive disease and disorders of water balance in the central nervous system.

INTRODUCTION

There is strong direct and indirect evidence that the mammalian cardiac atria are powerful endocrine organs involved in the control of blood pressure, extracellular fluid volume, and electrolyte balance. Cardiac atrial muscle cells are known to contain secretory-like storage granules which share light and electron microscopic properties with peptide-containing storage granules in the gut (De Bold, 1978, 1982b; De Bold et al., 1978; Jamieson and Palade, 1964; Kisch, 1953). The cardiac content of those granules was shown to depend on changes in the water

Department of Zoology, Section of Wildlife Research and Parasitology, University of A-8010 Graz, Graz, Austria.

2Clinical Neuroscience Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20894.

339

0272-4340/88/1200-0339506.00/0 ~ 1988 Plenum Publishing Corporation

340 Skofitsch and Jacobowitz

and electrolyte balance (De Bold, 1978, 1979, 1982b; Marie et al., 1976) and corticoid levels (Cantin and Hunt, 1973). The granules itself were found to contain renin-like activity (Cantin et al., 1982). Recently a family of natriuretic peptides has been isolated and purified from secretory-like storage granules of the cardiac atria and termed atrial natriuretic peptides (ANPs) (Atlas et al., 1984; Currie et al., 1984a; De Bold, 1982a; Grammer et al., 1983; Kangawa and Matsuo, 1984; Kangawa et al., 1984a; Napier et al., 1984; Thibault et al., 1983b; Trippodo et al., 1983). Common amino acid sequences of several of these peptides indicated that they may be derived from a common precursor, which has been shown by cloning of the human, rat, and mice complementary DNA of atrial prepro-ANP-messenger RNA that encode the precursor of those peptides termed ANPs (Atlas et al., 1984; Currie et al., 1984a; Flynn et al., 1983, 1985; Gardner et al., 1986; Greenberg et al., 1984; Kangawa and Matsuo, 1984; Kangawa et al., 1984a, b, 1985; Maki et al., 1984a, b; Misono et al., 1984; Miyata et al., 1985; Nakayama et al., 1984; Napier et al., 1984; Nehmer et al., 1984; Oikawa et al., 1984; Seidmann et al., 1984a, b; Sugiyama et al., 1984; Thibault et al., 1983a, 1984b; Trippodo et al., 1984a; Voulteenaho et al., 1985; Yamanaka et al., 1984; Zivin et al., 1984). It is likely that the high molecular weight forms of the ANPs are compartmented within the storage granules; they are cleaved enzymatically upon release to low molecular weight forms (Harris and Wilson, 1985; Trippodo et al., 1984a; Voulteenaho et al., 1985) which are measured in plasma by radioimmunoassay (Tanaka et al., 1984; Xie et al., 1986). ANP-like immunoreactivity (-ir) has been localized by immunocytochemistry and radioimmunossay in heart (Cantin et al., 1984; Gutkowska et al., 1984a, b; Sonnenberg et al., 1983; Tanaka et al., 1984; Tang 1984a), salivary glands (Cantin et al., 1984), the autonomic nervous system (Debinsky et al., 1986; Papka et al., 1985), the neurocardiac axis in snails (Nehls et al., 1985), and the central nervous system of frogs (Nechitailo et al., 1987) and mammals (Jacobowitz et al., 1985; Kawata et al., 1985; Morii et al., 1985; Saper et al., 1985; Skofitsch et al., 1985; Standaert et al., 1986a; Tanaka et al., 1984; Zamir et al., 1986). Binding sites of radiolabeled ANPs have been found in the thymus and spleen (Kurihara et al., 1986, 1987a), adrenal and kidney (Chai et al., 1987; Swithers e t a l . , 1987), brain microvessels (Chabrier et al., 1986, 1987), and central nervous system (Gibson et al., 1986; Kurihara et al., 1987b, McCarty and Plunkett, 1986a, b; Quirion et al., 1984; Saavedra et al., 1986a, b, c, 1987).

Several peripheral (hormonal) functions of the ANPs have been described: (a) diuretic and natriuretic activity (Borenstein et aL, 1983; De Bold, 1979; De Bold and Salerno, 1983; De Bold et al., 1981; Flynn et aL, 1983; Keeler, 1982; Keeler and Azzarrolo, 1983; Sagella and McGregor, 1984; Seymore et al., 1984; Thibault et aL, 1984a), which currently is believed to depend on (b) central dopaminergic activation (Maringrez et al., 1985; Pettersson et al., 1986; Webb et al., 1986); (c) vasorelaxant activity of the smooth muscle of blood vessels (Faison et al., 1985; Garcia et al., 1984a, b; Grammer et al., 1983; Kleinert et al., 1984; O'Donell et al., 1985; Winquist, 1985; Xie et al., 1986) and airways (O'Donell et al., 1985) and decrease in mean arterial blood pressure (Maak et al., 1984; Tang et al., 1984b); (d) inhibition of adenylate cyclase (Anad-Strivastava et al., 1984;

Atrial Natriuretic Peptide in Rat CNS 341

and (e) increase in aldosterone production (Atarashi et al., 1984; Cartier et al., 1984a, b; De Lean et al., 1984; Goodfriend et al., 1984).

The mechanism of the release of ANPs from cardiac atria also has been studied. ANPs are believed to be released from the cardiac atria by (a) mechanical stimuli [e.g., distension of the atria (Currie et al., 1984b; Katsube et al., 1985; Lang et al., 1985; Ledsome et al., 1985], (b) acute blood volume expansion [e.g., infusion of dextrose (Lang et al., 1985; Eskay et al., 1986; Zamir et al., 1986)], (c) hormonal stimuli [e.g., vasopressin (Katsube et al., 1985; Manning et al., 1985)] or activation of the renin-angiotensin system (Itoh et al., 1987) or hormones of an anterior pituitaryorigin (Zamir et al., 1987).

Although peripheral actions of ANP are well known, there is little known about ANP activity in the central nervous system. Immunocytochemical and receptor localization suggests that neuronal ANP may be involved in the central control of blood pressure and fluid and electrolyte balance (Chai et al., 1986; Kurihara et al., 1987b; McCarty and Plunkett, 1986a, b; Quirion et al., 1984; Swithers et al., 1987).

Rational approaches to the study of the function of central ANPs require the knowledge of the discrete localization of the peptides and their receptors in the central nervous system. In this we review detailed stereotaxic maps of the distribution of ANP-ir neurons and receptor binding sites in the rat brain using indirect immunofluorescence methods and in vitro autoradiographic techniques. A comparison of the localization of peptide and receptor binding sites is presented, in addition to quantitative values of ANP in discrete regions of the brain. High-performance liquid chromatography studies and cardiovascular dyna- mic studies are also reviewed.

ANTISERA

Specific antisera were raised in male New Zealand rabbits against tr-rat- ANP(5-28), also called atriopeptin III, coupled either to bovine thyroglobulin by carbodiimide (Goodfriend et al., 1969) or to human thyroglobulin by glutaraldehyde (O'Shanghnessy, 1982) using standard immunization procedures (Vaitu- kaitis et al., 1971). Bleeds were tested for indirect immunocytochemistry and radioimmunossay every 3 weeks.

For immunocytochemical purposes only antisera raised against c~-rat-ANP(5- 28) coupled to human thyroglobulin by glutaraldehyde were found to be useful, as they revealed comparatively less background and better staining than did those raised against the same peptide but coupled to bovine thyroglobulin by carbodiimide.

STEREOTAXIC ATLAS OF A N P DISTRIBUTION

A complete stereotaxic atlas showing the discrete localization of ANP-ir nerves and cell bodies was prepared according to Jacobowitz and Palkovits (1973)


and Palkovits and Jacobowitz (1973) by indirect immunofluorescence (Coons, 1958) with modifications to standard immunocytochemical procedures described in detail previously (Skofitsch and Jacobowitz, 1985a-c; Skofitsch et al., 1985). Stereotaxic coordinates were defined according to the atlases of K6nig and Klippel (1963) and Paxinos and Watson (1982). Untreated rats and rats pretreated with colchicine (i.c.v.) 2 days prior to the experiment were perfused via the heart with 10% formalin. The central nervous system was removed and frozen on dry ice. Cryostat sections were processed for indirect immunofluorescence.

The sections were incubated in specific antisera diluted 1:750 for 3 days followed by incubation in fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG diluted 1:400 for 30 min. Fluorescence was monitored with a Leitz Orthoplan fluorescence microscope equipped with a dark-field condenser and a Ploemopak illuminator.

As an immunohistochemical control the antiserum was preabsorbed with 10-6M synethetic tr-rat-ANP(5-28), which resulted in a complete loss of immunofluorescence. ANP-ir cell bodies were observed in colchicine-pretreated rats only; nerve fibers were observed in both colchicine-pretreated and untreated rats. A complete stereotaxic mapping of ANP-ir cell bodies and fibers is given in Figs. 1-15. A summary of areas and/or nerves is given in Table I. Cells and fibers are "semiquantitatively" rated as + (sparse), + (low), + + (moderate), + + + (dense), or + + + + (very dense).

Very dense accumulations of ANP-ir cell bodies were observed in the nuclei surrounding the most rostral portion of the third ventricle, the so-called area of the "anteroventral third ventricle" [AV3V (Brody et al., 1978; Hartle and Brody, 1984)], the organum vasculosum lamina terminalis (OVLT) (Figs. 4A, 16, 17), the medial preoptic nucleus (Figs. 4B and C, 17), the suprachiasmatic nucleus (Fig. 5), the preoptic periventricular nucleus (Figs. 4B-D, 17), and the ventral premamillary nucleus (Figs. 7D, 21A and B).

Dense accumulations of ANP-ir cell bodies were located in the hypothalamic periventricular nucleus (Figs. 6A and B, 20A, 21A), leaving the dorsal third nearly without cells except at the caudal aspects, where dense accumulations of ANP-ir cells were observed in the periventricular subdivisions of the hypothalamic paraventricular nucleus (Figs. 6A and B, 15, 18A, 19). Other areas with a dense appearance of cell bodies are the retrochiasmatic area (Figs. 6A and B), and the arcuate nucleus (Figs. 6D-7D, 20, 21A), the dorsomedial nucleus (Figs. 6D-7B), the subzona incerta (Figs. 6B-D, 19), the base and the surroundings of the medial mamillary body (Figs. 8A and B, 22), the most lateral aspects of the medial habenula (Figs. 7C and D, 21C and D), and the caudal extensions of the dorsal raphe nucleus lining the fourth ventricle just above the dorsal tegmental nucleus (Figs. 11, 24).

Moderate accumulations of ANP-ir cells were seen in the ventral part of the bed nucleus of the stria terminalis (Fig. 4A), the anterior and medial parvocellular parts of the hypothalamic paraventricular nucleus (Figs. 6A and B, 15, 18A, 19), the perifornical area within the medial forebrain bundle (Figs. 6D-7C, 20B), the caudal part of the medial mamillary body (Fig. 8C), and the lateral aspects of the dorsal raphe nucleus (Fig. 10C).


b,

13

Figs. 1-14. Schematical drawings of coronal sections of the rat brain and spinal cord according to Jacobowitz and Palkovits (1973) and Palkovits and Jacobowitz (1973). Coordinates are taken from the topographic atlas of K6nig and Klippel (1963) and Paxinos and Watson (1986) and given as micrometers anterior or posterior to the intraaural plane. The relative density and distribution of ANP-like immunoreactive neurons (cell bodies--asterisks; axons, fibers, and endings--small dots) are indicated on the left. The appearance of density of t25I-ol-ANP(5-28)-labeled binding sites in autoradiography is indicated with dots on the right.

Few A N P - i r cells were seen in the dorsa l aspects of the b e d nucleus of the str ia t e rmina l i s (Fig. 4), in the a r ea b e t w e e n the cen ta l and the med ia l a m y g d a l o i d nucleus (Fig. 7B) , the basa l h y p o t h a l a m u s dorsa l to the op t ic t rac t (Figs. 5 C - 6 C ) , the l a te ra l dorsa l t e g m e n t a l nucleus (Figs. l l A - C , 24), the dorsa l and vent ra l p a r a b r a c h i a l nucle i (Fig. 11A), the spinal nucleus of the fifth ne rve

A


Fig. 2

(Figs. 13A-D), the substantia gelatinosa of the fifth nerve (Fig. 13E), and the nucleus commissuralis (Fig. 13E).

Sparse and rarely ANP-ir cells were seen in the anterior hypothalamic nucleus (Figs. 5A-6A), the magnocellular part of the hypothalamic paraventricular nucleus (Figs. and 6A and B), the ventromedial nucleus (Figs. 6C-7B), the posterior hypothalamic nucleus (Figs. 7C-8B), the central gray matter (Figs. 8B-10D), the lateral part of the substantia nigra (Figs. 9A-C), the rostral and dorsal parts of the interpenduncular nucleus (Figs. 9A and B), the most caudal


0

0

Fig. 3

part of the locus coeruleus (Fig. l lD) and the lining of the fourth ventricle dorsal to the vestibular nucleus (Fig. 12).

Delicate varicose nerves containing ANP-ir were seen in both untreated and colchicine-treated rats and were widely distributed in the rat central nervous system.

A very dense network of ANP-ir fibers was seen in the organum vasculosum lamina terminalis (OVLT; Figs. 4A, 16, 17), the preoptic suprachiasmatic area (Figs. 4B-D, 17), the preoptic periventricular nucleus (Figs. 4B-D, 17), and the lateral and dorsal subdivisions of the interpeduncular nucleus (Figs. 9B and C, 23).


0

Fig. 4

Dense accumulations of ANP-ir fibers were observed in the ventral bed nucleus of the stria terminalis (Figs. 4A-C), the hypothalamic suprachiasmatic nucleus (Fig. 5), the ventral hypothalamus extending from the retrochiasmatic ~ e a (Figs. 6A and B), and the arcuate nucleus (Figs. 6C-7, 20A, 21A), dorsal in the preoptic commissure toward the hypothalamic medial forebrain bundle. All


0

Fig. 5

subdivisions of the hypothalamic paraventricular nucleus show dense fiber accumulations extending laterally via the tractus filiformis dorsal to the fornix and to the medial forebrain bundle (Figs. 6A-C, 19). Other areas with dense ANP-ir innervation are the subzona incerta (Figs. 6B-D), the ventcal premamillary nucleus (Figs. 7C and D, 19), the marginal aspects of the medial mamillary

348 Skofltsch and Jacobowitz

Fig. 6

nucleus (Figs. 8B, 22), and the lateral subdivision of the medial habenular nucleus (Figs. 7C and D, 21C and D).

A moderate innervation with ANP-ir nerves was seen in the ventral aspects of the central amygdaloid and the medial amygdaloid nuclei (Figs. 5, 6), the lateral septal nuclei (Figs. 3B-4B), the medial aspects of the accumbens nucleus

Atrial Natriuretic Peptide in Rat CNS

f

349

Fig. 7

(Fig. 3), the tractus septohypothalamicus and diagonalis (Figs. 3, 4A), the medial preoptic nucleus (Figs. 4B-D, 17), the external zone of the median eminence (Figs. 6C-7C, 18B, 21A), the dorsomedial (Figs. 6D-7B) and posterior hypothalamic nuclei (Figs. 7C and D), the periventricular thalamic, gelatinosus, supra- frascicularis, and rhomboid nuclei (Figs. 5B-7), the tegmental reticular nucleus of


Fig. 8

the pons (Fig. 10C), the dorsal and ventral parabrachial nuclei (Figs. l l A and B), the lateral dorsal tegmental nucleus (Figs. l l A - C , 24), the locus coeruleus (Fig. 11), and the caudal extensions of the dorsal raphe nucleus and the lining of the fourth ventricle (Figs. 10, 11, 12A, 24).

Few ANP-ir fibers were seen in the rostral medial cerebral cortex (Fig. 3A), the dorsal bed nucleus of the stria terminalis (Figs. 4A and B), the anterior hypothalamic nucleus (Figs. 5, 6A), the supraoptic nucleus (Figs. 4D, 5), the reuniens nucleus (Figs. 5D, 6), the area postrema (Fig. 13C), and the posterior pituitary.

A sparse appearance of ANP-ir fibers was noted in the amygdaloid complex


O0

Fig. 9

00

A900

except in the central and medial nuclei (Figs. 6, 7), the rostal medial forebrain bundle (Figs. 3-5), the lateral preoptic nucleus (Figs. 4B-D), the subfornical organ (Fig. 5C), the ventromedial nucleus (Figs. 6C and D, 7A and B), the central gray (Figs. 8B-10), the lateral nucleus of the substantia nigra and the medial lemniscus (Fig. 9), the medullary areas lining the fourth ventricle dorsal to the vestibular nuclei (Fig. 12), the borderline areas between the tract and the nucleus of the spinal trigeminal nerve extending caudally to the substantia gelatinosa (Fig. 13), and the superficial layers of the spinal cord (Fig. 14). In the


30

O0

500

'1000

Fig. 10

spinal cord fibers were occasionally seen around the central canal and in the ventral horn.

Recently, two other mappings of ANP in a detailed format have appeared (Kawata et al., 1985; Standaert et al., 1986a). Good agreement of the neuronal localization of ANP immunoreactive nerves was reported in those areas known to be involved in fluid and electrolyte balance (OVLT, preoptic-periventricular area), the parvocellular hypothalamic paraventricular nucleus, lateral hypothala-


Fig. 11

mic area, bed nucleus of the stria terminals, habenula, and interpeduncular nucleus. In addition, Kawata et al. (1985) have reported the localization of ANP-containing fibers in the glomerula and olfactory nerve layer of the olfactory bulb. A variety of differences, however, was noted among the three groups. Kawata et al. (1985) reported the presence of cell bodies in the endopiriform nucleus, cingulate cortex, medial amygdaloid nucleus, and interpeduncular


D

E . 000

" hJ JP5500 Fig. 12

nucleus, which was not supported by the work of Skofitsch et al. (1985), Standaert et al. (1986a), and Hamill et al. (1986). Standaert et al. (1986a) reported the presence of cell bodies in the central amygdaloid nucleus, pedunculopontine tegmental nucleus, ventrolateral medulla, and nucleus of the solitary tract which were not noted by Skofitsch et al. (1985) and Kawata et al. (1985). Skofitsch et al. (1985) did not report cells in the septal area which were mapped by Kawata et al. (1985) and Standaert et al. (1986a).

A particularly interesting group of ANP-immunoreactive cells contained in the laterodorsal tegmental and the pedunculopontine nuclei has been shown to


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Fig. 13 C) 0

coexist with choline acetyltransferase-like immunoreactivity (Standaert et al., 1986b). A subpopulation of the ch01inergic neurons in the laterodorsal tegmental nucleus was previously reported to contain substance P and corticotropin releasing factor (Crawley et al., 1985, 1986). The significance of the ANP- cholinergic cell body coexistence requires further study. Whether or not ANP in these cholinergic somata is also contained in the terminal projection sites is open to question. A known cholinergic projection site from the laterodorsal tegmental nucleus is the anteroventral nucleus of the thalamus (Rotter and Jacobowitz, 1981). It is noteable that ANP-immunoreactive fibers were not seen in the anterovental nucleus of the thalamus (Skofitsch et al., 1985; Kawata et al. , 1985; Standaert et al., 1986a). This is reminiscent of the peptide galanin which is observed within the cell bodies of the locus coeruleus but not throughout the cortical projection sites (Skofitsch and Jacobowitz, 1985b).


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5

5 Fig. 14

Table I. Distribution of ANP-ir and ANP-Binding Sites in Various Regions of the Rat Brain

Rating ANP-ir Map, Fig. ( fmol /mg Binding

Receptor region No.(s) Cells Fibers protein) sites

Telencephalon Bulbus olfactorius _ + +a (n .m.) + + + Tractus olfactorius lateralis - +a (n .m.) + + + Tuberculum olfactorium _ +a (69.9 + 3.7) + Frontal cortex 1, 2 - + + (47.4 5: 2.6) Cingulate cortex 2 -4 +a + +a (46.0 ± 3.0) + + Hippocampus 6 -8 +" + +~ (38.5 + 1.5) + + N. tractus diagonalis 3 C - 4 A - + + (108.0 4- 7.0) N. caudatus 2 C - 7 - +" (35.2 + 2.2) N. amygdaloideus centralis 6 -7 + + + (87.0 + 6.3) N. amygdaloideus medialis 6 -7 + + + (90.7 ~: 5.6) N. interstitialis stria terminalis

pars dorsalis 4 + + (126.0 + 17.0) + + N. interstitialis stria terminalis

pars ventralis 4 + + + + + (246.0 + 22.6) Diencephalon

N. preopticus periventricularis 4 + + + + + + + + (464.4 ± 40.7) N. preopticus medialis 4 + + + + + (558.0 + 64.4) N. suprachiasmaticus 4 + + + + + + + + (207.2 + 13.3) + + N. periventricularis hypothalami 5 -7 + + + + + + (390.7 + 39.6) N. hypothalamicus anterior 5 - 6 A + + (278.7 :t: 23.7) N. supraopticus 4 D - 6 A - + (97.3 + 15.6) + + N. paraventricularis 6A, B + + + + + + (580.9 + 37.7) + + + + b MFB; medial forebrain bundle 4 -8 + + + + + (152.1 + 12.6) +


Table L (Continued)

357

Rating ANP-ir Map, Fig. (fmol/mg Binding

Receptor region No.(s) Cells Fibers protein) sites

N. dorsomedialis 6D-7B + + + + + (194.6+ 15.2) N. ventromedialis 6C-7B ± ± (130.2 ± 17.0) N. arcuatus 6C-7 + + + + + + (255.3±22.2) + + + + Eminentia mediana 6C-7 - + + + (460.7 ± 53.3) + + + + N. hypothalamicus posterior 7C-8A + + + (146.5 ± 15.5) N. mamillaris medialis 8 + + + + + + (60.7±4.8) N. periventricularis thalami 5-7 - + + (237.2 ± 9.3) N. habenulae medialis 6-7 + + + + + + (235.0+20.4) + + + +

Mesencephalon Substantia nigra p. reticularis 8B-9C - - (44.1 ± 6.3) N. interpeduncularis 9 ± + + + + (468.8 ± 27.8) + + + + Colliculus inferior 10B-11A - - (35.9 ± 3.3) Substantia grisea centralis 8B-10 ± ± (209.8 ± 28.5) N. raphe dorsalis p. rostralis 9C- i0 + + ± (182.0 ± 26.3) N. raphe dorsalis p. caudalis 11 + + ± (96.9 ±4.1) + + + Lemniscus medialis 8-10 - ± (40.2 ± 8.2)

Pons Locus coeruleus 11 ± + + (143.9 ± 15.5) + + + N. parabrachialis dorsalis l l A - 1 1 C ± + + (125.4 + 5.9) N. tegmenti dorsalis 11 - + (99.5 5: 5.2) N. tegmenti dorsalis lateralis 11 - + + (157.3 ± 14.1)

Cerebellum Cerebellum 11 - - (24.4 ± 2.6) +

Myelencephalon Tractus spinalis nervi trigemini 12, 13 + + (29.3 ± 1.6) N.t. spinalis nervi trigemini 12, 13 ± + (29.2 ± 2.2) N. solitarii 12D-13D - + (69.2 ± 7.0) + + +

Medulla spinalis Cornu dorsalis 14 - + (38.1 ± 2.6) + + Cornu ventralis 14 - + (37.4 ± 2.2) +

Hypophysis Neurohypophysis - - - + (30.7 ± 2.2) Adenohypophysis - - - - n.d. + + + +

Circumventricular organs O. vasculosum lamina terminalis 4A + + + + + + + + (320.1 ± 52.9) + + + + Organum subfornicale 5C, D - + (86.6 ± 6.3) + + + + Area postrema 13C, D - + (54.8 + 5.2) + + + +

a See Kawata et al. (1985). b See Kurihara et al. (1987).

RADIOIMMUNOLOGICAL IDENTIFICATION AND QUANTIFICATION

U s i n g a n a n t i s e r u m p r o d u c e d in r a b b i t s a g a i n s t o l - r a t - A N P ( 5 - 2 8 ) c o u p l e d to

b o v i n e t h r y o g l o b u l i n b y c a r b o d i i m i d e ( G o o d f r i e n d et al . , 1964) a n d t h e p r e p a r a -

t i o n o f 125I -o~- ra t -ANP(5-28) ( H u n t e r a n d G r e e n w o o d , 1962) , a s e n s i t i v e d o u b l e -

a n t i b o d y r a d i o i m m u n o a s s a y ( R I A ) w a s d e v e l o p e d , t h u s a l l o w i n g q u a n t i t a t i o n o f

t i s sue l e v e l s o f A N P - i r a n d b i o c h e m i c a l c h a r a c t e r i z a t i o n o f b i o l o g i c a l l y a c t i v e

f o r m s o f A N P s t o r e d in a n d r e l e a s e d f r o m n e r v o u s t i s s u e ( Z a m i r et al . , 1986) .

R a d i o i m m u n o a s s a y . D i s e q u i l i b r i u m R I A w a s p e r f o r m e d in a 5 0 m M


\ ap~ ~ . #_,ve~ pm rnp~ V

am

@

Fig. 15. Schematic drawings of different levels of the nucleus paraventricularis and its subdivisions modified after Sawchenko and Swanson (1982). The asterisks indicate the distribution of ANP-ir cell bodies. F, fornix; re, nucleus reuniens; npe, nucleus periventricularis hypothalami; V, third ventricle. Subdivisions of the nucleus paraventricularis: am, pars magnocellularis anterior; dp, pars parvocellularis dorsalis; lp, pars parvocellolaris lateralis; mp, pars parvocellularis medialis; pm, pars magnocellularis posterior.

sodium phosphate buffer at pH 7.6 containing 0.1% Triton X-100, 0.1% gelatin, 0.1% bovine serum albumin, and 0.01% merthiolate. The incubation volume was 500 #1 and consisted of 300/4 assay buffer containing standards or unknown, 100/~1 ANP-antiserum diluted 1:10,000 in assay buffer containing 2% normal rabbit serum, and 100/~1 12SI-labeled 0:-rat-ANP(5-28) diluted in assay buffer to about 6000 cpm/100/~1. Reagents were added to 12 x 75-mm polystyrene tubes, vortexed, and kept at 4°C for 1-6-24 hr. Thereafter 100/~1 of the second antibody (goat anti-rabbit y-globulin) diluted 1 : 20 in assay buffer was added and incubated at 4°C for another 16-24hr to form precipates, which were collected by centrifugation at 2000 g for 20 min. Supernatants were discarded, and the pellets counted in a gamma counter. The sensitivity of the assay was limited to 5 fmol ANP-ir; the EDs0 was 40-50 fmol m1-1.

In this RIA the antiserum fully recognized tr-rat-ANP(5-28), cr-rat-ANP(4- 28), a~-rat-ANP(3-28), 0:-rat-ANP(5-27), and a~-rat-ANP(5-25); only 10% cross-


Fig. 16. Fluorescence photomicrograph showing ANP-ir cell bodies and fibers in the OVLT (close to the bottom) and nucleus preopticus suprachiasmaticus (top portion) in the brain of a colchicine- treated rat (level A 7470). Bar represents 50/~m.

reactivity was observed toward 0L-rat-ANP(1-28); about 2% cross-reactivity was seen toward cr-human-ANP(1-28); and the antiserum did not cross-react with the following peptides---dynorphin B, Leu-enkephalin, /3-endorphin, angiotensin II, oxytocin, and Arg-vasopressin.

Characterization by RIA and HPLC. Male Sprague Dawley rats, weighing 200-250g, were killed by decapitation, the preoptic-hypothalamic tissue was dissected out, homogenized in a 10-fold amount of boiling 0.1 N HC1 for 10 min, then homogenized by sonication, and a 20-/~1 aliquot was taken for protein determination (Lowry et al., 1951). After centrifugation at 10,000 g for 30 min the supernatant was aliquoted and evaporated to dryness in a vacuum centrifuge.

An aliquot was rehydrated in RIA buffer and diluted in consecutive 1 : 1 steps for parallel displacement studies in RIA. Increasing concentrations of tissue extract (referred to as equivalents of /~g protein in Fig. 25) of the preoptic- hypothalamic area displaced radioactive ligand from the antiserum in a dose- dependent manner and in parallel to increasing concentrations of synthetic a~-rat-ANP(5-28), indicating a close relationship or even identity of preoptic- hypothalamic ANP-ir to the synthetic peptide.

Another aliquot of the preoptic-hypothalamic tissue extract was resuspended in water containing 0.1% trifluoroacetic acid and separated by high-performance liquid chromatography (HPLC). HPLC was performed using an Altex Ultrosphere-ODS reverse-phase column (5-gm particle size, 4.6 x 250 mm). The


Fig. 17. Fluorescence photomicrograph showing ANP-ir cell bodies and fibers in the organum vasculosum lamina terminalis (bottom, close to the chiasma opticum; CO) and the nucleus preoptieus suprachiasmaticus (above the OVLT) in a colchicine-treated rat (level A 7190). Bar represents 50 ~m.

elution medium was a linear gradient of acetonitrile (25-55%) in water containing 0.1% trifluoroacetic acid at a flow rate of 1 mlmin -1. One-minute fractions were collected, evaporated to dryness, and subjected to RIA. the HPLC-RIA profile of rat preoptic-hypothalamic tissue extract revealed two peaks. The major portion coeluted with synthetic cr-rat-ANP(5-28); the second peak eluted at acetonitrile concentrations much greater than the ANP-fragments tested and remains unidentified (Fig. 26).

Quantification. Male Sprague Dawley rats were killed by decapitation, and the brain was rapidly removed, frozen on dry ice, and cut into 300-/tm slices in a cryostat. Discrete brain areas were microdissected by the method of Palkovits (1973), transferred to 0.1NHC1, boiled for 10 min, then sonicated, and a 20-/~1 aliquot was removed for protein assay (Lowry et al., 1951). The remaining sample was centrifuged at 2000g for 20 min, and the supernatants were transferred to polystyrene tubes, evaporated to dryness in a vacuum centrifuge , and subjected to RIA.

The quantitative distribution of ANP-ir in discrete areas of the rat brain is


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Fig. 18. Fluorescence photomicrographs showing ANP-ir cells in the periventricular part of the nucleus paraventricularis of the hypothalamus of a colchicine-treated rat (upper panel; level A 5340), which corresponds to the schematic drawing in Fig. 15C, and the median eminence (lower panel) of an untreated rat with ANP-ir nerves in the external zone (level 4110). Bar represents 50/~m.

presented in Table I. Perusal of these values reveals that ANP-ir is unevenly distributed in individual nuclei and areas throughout the rat CNS.

The highest concentrations of ANP-ir ( > 4 0 0 f m o l m g -1 protein) were observed in the preoptic periventricular and medial preoptic nucleus, the hypothalamic paraventricular nucleus; the median eminence, and the interpeduncular nucleus.

High concentrations ( 2 0 0 - 4 0 0 f m o l m g -1 protein) were observed in the OVLT, the ventral bed nucleus of the stria terminalis, the thalamic periventricular nucleus, the habenula, the preoptic suprachiasmatic nucleus, the anterior hypothalamic and hypothalamic periventricular nuclei, the arcuate nucleus, and the central gray matter .

Moderate concentrations of ANP-ir ( 8 0 - 2 0 0 f m o l m g -1 protein) were observed in the subfornical organ, the nucleus of the diagonal band, the dorsal bed nucleus of the stria terminalis, the central and medial amygdaloid nuclei, the supraoptic nucleus, the medial forebrain bundle, the dorsomedial and ventrome-


Fig. 19. Fluorescence photomicrograph showing ANP-ir cells in the nucleus paraventricularis hypothalami and the subzona incerta (zi) of a colchicine-treated rat (level A 5000) which corresponds to the schematic drawing in Fig. 15D. Bar represents 50/~m.

dial nuclei, the posterior hypothalamic nucleus, the rostral part of the dorsal raphe, the locus coeruleus, the parabrachial nuclei, and the dorsal and dor- solateral tegmental nuclei.

Low concentrations (<80 fmol mg -1 protein) were observed in the frontal and cingulate cortices, the hippocampus, the olfactory tubercle, the caudate nucleus, the reticular part of the substantia nigra, the medial lemniscus, the inferior colliculus, the cerebellum, the tractus and nucleus of the spinal trigeminal nerve, the area postrema, the nucleus of the solitary tract, the dorsal and ventral spinal cord, and the neurointermediate lobe of the pituitary.

A comparison of these quantitative values with "semiquantitative" estima- tions of densities of nerve fibers (Table I) reveals, with few exceptions, a good correlation between the varicose fiber density and RIA values.

RECEPTOR AUTORADIOGRAPHY

Using autoradiographic techniques and (3-125I-iodotyrosy128)rat-ANp(28) (Amersham Labs, Amsterdam, Netherlands), receptor binding sites have been demonstrated throughout the rat CNS and adjacent structures. From the very beginning of neuropeptide autoradiography it has become apparent that neuropeptide receptor binding sites are not always located in close proximity to peptide-containing neuronal fibers or terminal fields (Jacobowitz and Skofitsch, 1986; Leroux and Pelletier, 1984; Shultz et al., 1984; Wynn et al., 1984). Such


Fig. 20. Fluorescence photomicrograph showing ANP-ir cell bodies and nerve fibers in the brain of a colchicine treated rat (A) in the nucleus periventricularis hypothalami adjacent to the third ventricle (V) and the nucleus arcuatus (na; level A4890) and (B) surrounding the fornix (F) and above the fornix in the nucleus perifornicalis (level A 4380). Bar represents 50/~m.

"mismatches" are the subject of considerable speculation. Nevertheless, autoradiography and topographical localization of neuropeptide binding sites are an important dimension that allows us to demonstrate potential sites of action of neuropeptides in brain.

The methods of receptor autoradiography for iodinated peptides that we have used in this paper are described in detail elsewhere (Skofitsch and Jacobowitz, 1985d; Skofitsch et al., 1986). Briefly, male Sprague Dawley rats, weighing 200 to 250 g, were perfused via the ascending aorta with 150 ml of ice-cold phosphate-buffered saline, and the brain and the cervical spinal cord were removed quickly, cut into 8-mm slices, frozen on dry ice, and cut into serial coronal 20-#m slices in a cryostat. The sections were thaw mounted on chrom-alum-coated slides, dried under a stream of cold, dry air, and frozen at -20°C until they were used for autoradiography.

Binding parameters have been widely published by others (Kurihara et al., 1987; Quirion et al., 1984; Saavedra et al., 1986a). Thus we used 100pM


Fig. 21, Fluorescence photomicrographs showing ANP-ir cells and nerve fibers in the brain of a colchicine-treated rat: (A) ANP-ir cells and fibers in the nucleus premamillaris ventralis (npmv; enlargement in B), the nucleus arcuatus (na), and the nucleus periventricularis hypothalami adjacent to the third ventricle (V; level A3430) and (C) in the nucleus habenulae mediafis (hm; enlargement in D, level A 3750) at the border to the nucleus habeulae lateralis (hi). Bar represents 50 ~m.

(3-~2sI-iodotyrosy128)rat-ANP(28) in the absence or presence of I0 #M unlabeled a~-rat-ANP(5-28) for incubation. Incubation time of sections in the labeled peptide was 45 min.

For autoradiography sections were preincubated in 50 mM Tris-HC1 buffer (pH7.7) containing 5mMMgCI2 and 2 m M E G T A to displace endogenous ligand. Thereafter they were incubated in the same medium but also containing 0.5% bovine serum albumin, 0.5% gelatin, 20,000KIU Trasylol (gift from Bayer--Austria, Vienna) in 50 ml buffer, and (3-12sI-iodotryrosy128)rat-ANP(28) to reach a final concentration of 33,000 cpm in 100/~1. For control purposes also 10 #M 0~-rat-ANP(5-28) was added, which resulted in a complete loss of binding sites in all areas investigated. Autoradiograms were developed using LKB 3H-sensitive film and exposure times of 9 to 16 days.

Representative autoradiograms of different brain areas are shown in Figs. 27-30. A summary and comparison of ANP-ir neurons with immunohistochemical and RIA data are given in Table I.

ANP-binding sites were found to be distributed in discrete regions of the rat brain. Binding sites were found in areas previously described by others (Kurihara et al. , 1987; McCarty and Plunkett, 1986a, b; Quirion et al. , 1984; Saavedra et al. , 1986a). However, we extended the work to the whole brain, giving a stereotaxic atlas of ANP-binding sites.

Very dense binding sites were observed in the meninges as well as in the ependymal cells lining the third ventricle, in the subfornical organ (Figs. 27G and H), the anterior and posterior choroid plexi (Figs. 27G, 29F and G), the arcuate


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Fig. 24. Fluorescence photomicrograph showing ANP-ir cells and fibers in the most caudal aspects of the nucleus raphe dorsalis (lateroventrally to the fourth ventricle; V), in the nucleus tegmenti dorsalis lateralis (ntdl), and lining the fourth ventricle; the nucleus tegmenti dorsalis (ntdl) is devoid of ANP-ir cells (level P 3000). Bar represents 50/~m.

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Displacement of 125I-o:-rat ANP by increasing concentrations of synthetic o:-rat ANP(5-28) (filled circles) or extracts of hypothalamic-preoptic tissue (open circles).

nucleus (Figs. 28B-D), the median eminence (Fig. 28B), the area postrema (Fig. 29H), and the anterior pituitary (Fig. 30C). A continuity of binding sites was followed from the medial habenula (Figs. 27H, 28A-E) through the fasciculus retroflexus (Figs. 28E-H) to the interpeduncular nucleus (Figs. 29A-C).

Dense ANP-binding sites were observed in the internal plexiform layer of the olfactory bulb, the lateral olfactory tract (Figs. 27A-E), the anterior commissure (Figs. 27A-G), the anterior corpus callosum extending caudally (Figs. 27A- 29A), the forceps major of the corpus callosum, the locus coeruleus (Fig. 29E), an area ventral to the midregion of the fourth ventricle at the level of the genu of the seventh nerve (Fig. 29E), which might be a very caudal extension of the dorsal raphe nucleus (compare with Figs. 11D, 24), the nucleus of the solitary tract (Fig. 29H), and the nucleus commissuralis (Fig. 30A) as well as the nucleus of the 10th nerve (Fig. 29H).

Moderate binding sites were observed in layer III of the cingulate and frontal cortex area 2 (Figs. 27A-28H), the agranular retrosplenial cortex (Figs. 29A-C), the intermediate lateral septal nucleus (Figs. 27B-F), the septofimbrial nucleus (Fig. 27G), the dorsal bed nucleus of the stria terminalis (Figs. 27E and F), the fimbria hippocampi (Fig. 27H), ammons horn areas CA1, CA3, and CA4 (Figs. 28B-29A), the subiculum (Figs. 28G and H), the granular layer of the dentate gyrus at the caudal level (Fig. 29B), the reticular thalamic nucleus (Figs. 28B-E),


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the thalamic stria medullaris (Figs. 27F-28A), the stria terminalis (Figs. 28B-E), the internal capsule (Figs. 27G-28G), the ansa lenticularis, the fornix (Figs. 27F-H), and the suprachiasmatic nucleus (Fig. 27H).

Low densities of ANP-binding sites were observed in the medial forebrain bundle (Figs. 27E-28E), the lateroventral hypothalamic area (Figs. 27F-28E), the reticular pontine and medullary areas (Figs. 29-30A), the granular layer of the cerebellum (Figs. 29E-30A), and the dorsal horn of the spinal cord (Fig. 30B); the ventral horn had a low to moderate appearance of ANP-binding sites, as has the neurointermediate lobe of the pituitary (Fig. 30C).

It is noteworthy that besides the OVLT, which is densely labeled by receptor autoradiography and immunocytochemistry, showing a very dense appearance of ANP-ir cell bodies and nerves, the second highest structure labeled by receptor autoradiography is the medial habenular nucleus, especially its lateral parts, the fasciculus retroflexus and the interpeduncular nucleus. In immunocytochemistry also there are dense accumulations of ANP-ir cells and fibers in the lateral aspects of the medial habenular nucleus and a very dense accumulation of nerve fibers in the dorsomedial part of the interpeduncular nucleus. Fibers seem to descend from the medial habenula accompanying the fasciculus retroflexus and the ventral tegmental area to the interpeduncular nucleus. Thus the habenular-


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Fig. 27. (A) A 10050; (B) A 8920; (C) A 7890; (D) A 7470; (E) A 7250; (F) A 6860; (G) A 6750; (H) A 6060.

in terpeduncular connect ion is densely labeled in immunoreac t iv i ty and receptor au toradiography; however , its funct ion is not clear yet.

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solitary tract and the commisuralis and vagal nucleus suggests modulation of the baroreceptor reflex loop.

A comparison of the distribution of ANP binding sites to the localization of ANP-ir fibers revealed the following. (a) A good correlation of high-density immunoreactive fibers and binding sites was observed in the OVLT, medial habenular nucleus, interpeduncular nucleus, paraventricular nucleus (Kurihara et al., 1987), and arcuate nucleus. (b) High-density binding sites correlated with a moderate density of ANP fibers in the olfactory bulb [see Kawata et al. (1985) for


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fiber density] and the caudal portion of the locus coerulus. (c) High-density binding sites in the subfornical organ and area postrema showed a poor correlation with low-density nerve fibers. High-density binding sites were observed in the anterior pituitary lobe, which was devoid of nerves. (d) Moderate-density binding sites were observed in the cingulate cortex, which was devoid of immunoreactive fibers. It should be pointed out, however, that Kawata


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et al. (1985) reported the presence of immunoreactive fibers and cell bodies in the cingulate cortex.

C A R D I O V A S C U L A R A C T I O N OF A N P

Recently, it has been demonstrated that microinjections of neuropeptides into different preoptic and hypothalamic nuclei of the anesthetized rat result in long-lasting cardiovascular effects (Diz and Jacobowitz, 1983, 1984a-d; Diz et al., 1984; Sills et al., 1985).

Systemic administration of ANPs has been shown previously to exhibit powerful vasorelaxant activity (Faison et al., 1985; Garcia et al., 1984a, b; Grammer et al., 1983; Kleinert et al., 1984; O'Donell et al., 1985; Winquist, 1985; Xie et al., 1986); furthermore, ANP-ir has been shown by immunohistochemistry and RIA to be localized mainly in areas of the CNS, e.g., the AV3V area, known to be essential for central blood-pressure control and fluid and electrolyte balance (Brody et al., 1978), drinking behavior, and angiotensin-induced central pressor responses (Buggy and Fisher, 1976; Buggy and Johnson, 1977; Buggy et al., 1977). Thus attention has been focused on cardiovascular effects of ANP following microinjections into the AV3V area of the rat (Sills et al., 1985).

Briefly, male Sprague Dawley rats weighing 250 to 350 g were used under halothane anesthesia. The femoral artery was cannulated for blood-pressure and heart-rate recordings. The animals were positioned in a Kopf sterotaxic ap- paratus, and a double-barreled glass micropipette with a tip o.d. of 20 to 75 #m was lowered into the brain at an angle of 4 ° to caudal to the final sterotaxic coordinates: 0.6 mm lateral to the midline, 0.3 mm anterior to the bregma, and 8.1 mm below the dura mater. One barrel was filled with PBS; the other one contained oL-rat-ANP(5-28) in PBS. After a 30-min equilibration period, animals were preinjected with PBS to recognize effects of the fluid volume (50 to 100 nl)


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or PBS vehicle. After another 30 min 2 to 40 pmol of peptide was injected. The injection site was localized in the anterior portion of the preoptic suprachiasmatic nucleus (POSC) at the very rostral end of the third ventricle, where high values of ANP-ir fibers were measured by RIA (Zamir et al., 1986; Kawata et al., 1985). The injection of 2 to 40 pmol of cr-rat-ANP(5-28) resulted in a dose-dependent elevation of systolic blood pressure and pulse pressure, but not diastolic or mean arterial blood pressure, and a dose-dependent increase in heart rate. The response to ANP injection is characterized by a delayed-onset (15 to 45 min) but long-lasting (2 to 4hr) effect (Fig. 31). After repeated injections of ANP, tachyphylaxis occurred.

The data are of special interest since central and peripheral ANP injection have opposite effects. In general, peripheral cardiovascular administration of ANP causes vasorelaxation, a drop in blood pressure, and bradycardia, with an almost immediate onset of effects and moderate duration; intracerebral applica- tion, however, so far concerning the POSC causes only vasoconstriction, an increase in blood pressure, and a pronounced tachycardia, with a slow and delayed onset and a very long-lasting effect.

At present, however, the mechanism of centrally induced effects of ANP injection into the POSC is unclear. A possibility is the direct activation of the sympathetic nervous system, however, further studies are required to elucidate the central effects of ANP on cardiovascular function.

DISCUSSION

From a functional standpoint it is noteworthy that receptor binding sites and ANP-ir are observed in an area known as the "anteroventral third ventricle" (AV3V). The AV3V includes the preoptic periventricular nucleus, the median preoptic nucleus, and the anterior wall of the third ventricle with the associated


OVLT (Brody et al., 1978). The AV3V region has been described as a critical area for the development and maintenance of experimental hypertension (Hartle and Brody, 1984). This area is also considered important in fluid and electrolyte balance. It appears that only the OVLT contains dense binding sites and that there are apparent mismatches between ANP-ir fibers (supported by high RIA values for the peptides) and receptor binding sites in most of the above-named AV3V regions. The significance of mismatches between neuronal varicosities and receptor binding sites is currently a matter of speculation. The absence of histochemically demonstrable terminal fibers may be merely a reflection of depletion of the peptide due to high turnover. The opposite situation, i.e., low or no binding sites in regions containing dense varicose fibers (e.g., preoptic periventricular nucleus, median preoptic nucleus), is even more perplexing.

Regulation o f Blood Pressure. A study designed to investigate possible cardiovascular effects following microinjection of ANP into the preoptic suprachiasmatic nucleus (POSC) revealed significant increases in heart rate and blood pressure (Fig. 31). The POSC is an AV3V region ventral to the medial preoptic nucleus. It is noteworthy that the onset of effects produced by ANP on blood pressure and heart rate was seen 15-45 min after injection. Peak effects were usually observed approximately 60-150 min after onset, and the duration of the effect was 2-4 hr, after which time values usually returned to baseline levels (Sills et al., 1985). Whether such a biological response can occur in a region of low density of receptor binding sites needs further study. However, it cannot be ruled out that diffusion of ANP occurred rostrally to stimulate OVLT receptors or caudally to influence suprachiasmatic nucleus receptors. Such diffusion may explain the long onset of action.

Another area important for control of blood pressure is the nucleus of the solitary tract (nts) and the motor nucleus of the X (vagal) nerve (nX), which contains dense receptor binding sites. These are areas that process baro- and chemoreceptor information.

In adult spontaneously hypertensive rats, Saavedra et al. (1986c) reported that the number of ANP binding sites was decreased in the nts. Furthermore, in the hypothalamus and pons, the ANP content was significantly increased in spontaneously hypertensive rats as compared to Wistar-Kyoto controls (Imada et al., 1985). The hypothalamus, pons, and plasma ANP concentration was increased in the high-salt group of Dahl salt-sensitive rats, which developed hypertension, in contrast to the salt-resistant rats, which were normotensive (Tanaka and Inagami, 1986).

In addition, Shimizu et al. (1986) demonstrated that the hypertension caused by an intraventricular injection of angiotensin II was attenuated by the concurrent injection of ANP. Itoh et al. (1986) also concluded that the exaggerated salt appetite observed in spontaneously hypertensive rats is blunted by centrally administered ANP, thereby supporting the notion that brain ANP plays a role in water and electrolyte homeostasis and in blood-pressure control.

Regulation of Fluid Balance. The subfornical organ (SFO) is one of the heaviest ANP-binding sites of the body. The SFO is a small circumventricular structure located in the roof of the third ventricle and attached to the ventral


surface of the hippocampal commissure. A variety of studies has demonstrated that the SFO plays a regulatory role in both fluid and sodium balance (Dellmann and Simpson, 1979; Simpson, 1981). Another dense binding circumventricular organ is the area postrema. The SFO, OVLT, and area postrema are outside the blood-brain barrier. Thus plasma ANP, derived from the heart, may act on the brain via these circumventricular organs. Brattleboro rats with diabetes inspidus showed a significant increase in the maximum binding capacity (Bmax) and affinity constant (Ka) for 125I-ANP-28 in the SFO, which is consistent with an up- regulation of ANP (McCarty and Plunkett, 1986b; Saavedra et al., 1986b). No changes, however, were noted in the area postrema (McCarty and Plunkett, 1986b). This is thought to represent a compensatory response to the profound disturbance in body fluid homeostasis which is a result of the absence of vasopressin. Increased ANP binding sites were also observed in the SFO and choroid plexus after 4days of water deprivation, thereby providing further support for a central role for ANP in the regulation of water balance (Saavedra et al., 1987).

Diuretic and Natriuretic Effect. Several studies have appeared that indicate that ANP has a variety of actions coordinated to normalize extracellular fluid volume. The diuretic and natriuretic action of ANP observed in the periphery appears to be, at least in part, centrally mediated. Intracerebroventricular administration of ANP(6-33) to conscious hydrated or salt-loaded rats resulted in a significant increase in urinary volume and sodium excretion (Israel and Barbella, 1986). Furthermore, infusion of ANP into the lateral ventricles stimulated urine flow in both normally hydrated and sodium-depleted conscious rats (Fitts et al., 1985). ANP also reduced salt appetite in rats following depletion of sodium by combined treatment with furosemide diuresis and a low-sodium diet. It was also found that centrally administered ANP blocked saline preference in sodium-depleted rats (Antunes-Rodrigues et al., 1986) and water deprivation decreased the content of ANP immunoreactivity in the neural lobe of the pituitary (Samson, 1985). In addition, the hypothalamic content of both vasopressin and ANP was significantly higher in rats fed a high-sodium diet than in the control group (Takahashi et al., 1986). It was suggested that the increased hypothalamic ANP is involved in the suppression of the vasopressin release in order to diminish their sodium appetite.

Antidipsogenic Effect. Antunes-Rodrigues et al. (1985) suggested that ANP released from the heart might act centrally to inhibit water intake by an action at one or more of the circumventricular organs. They demonstrated that intracerebroventricular and intravenous infusion of ANP in conscious overnight- dehydrated rats significantly inhibited subsequent water intake over a 2-hr test period. Their work further suggests that ANP opposes the action of the water-conservatory peptides angiotensin II (AII) and vasopressin. Water intake induced by central infusion of AII in rats was significantly inhibited by infusion of ANP (Antunes-Rodrigues et aL, 1985; Lappe et al., 1986; Nakamura et al., 1985, 1986). However, intravenous infusion of ANP failed to alter the dipsogenic action of centrally administered AII, which indicates that ANP found within the brain, but not the peripheral circulation, may participate in the regulation of extracellu-


lar fluid volume by modulating the dipsogenic actions of the central renin- angiotensin system (Lappe et al., 1986). Interestingly, ANP did not affect All-induced pressor responses (Nakamura et al., 1986).

The dipsogenic response to infused AII has been shown to be altered in rats with lesions in the SFO and OVLT (Bealer et al., 1979; Buggy et al., 1978; Lind et al., 1984; Thrasher et al., 1982). Al l and vasopressin have been considered to be cellular messengers in the central control of water intake. The interactive effects of these peptides is thought to explain, at least in part, the mechanism of thirst. There are several studies that support the idea that the central hormonal action of ANP is to oppose those of the water-conservatory peptides All and vasopressin and interface with the mechanisms of thirst. Central infusion of ANP results in significant reductions in basal arginine-vasopressin release (Samson and Eskay, 1986). Furthermore, the intracerebroventricular injection of ANP abol- ished the vasopressin release induced by the intraventricular injection of AII (Takahashi et al., 1986). It appears that ANP acts directly on Arg-vasopressin- producing cells, an idea which is supported by the dense number of binding sites in the paraventricular nucleus (Kurihara et al., 1987b) and immunoreactive cells and fibers (Jacobowitz et al., 1986; Kawata et al., 1985; Skofitsch et al., 1985; Standaert et al., 1986a).

It was shown that ANP added to the medium of a hypothalamus-posterior pituitary organ culture had no effect on basal vasopressin release but suppressed osmotically stimulated vasopressin secretion (Januszewiz et al., 1986). However, in a superfused rat pituitary gland, ANP significantly inhibited basal as well as All-stimulated vasopressin secretion (Obana et al., 1985). Similarly, in hypothal- amoneurohypophyseal explants and isolated neurointermediate lobes, ANP inhibited vasopressin release'(Crandall and Gregg, 1986). In addition, in v ivo and in vitro, ANP inhibited the release of both vasopressin and oxytocin when basal levels were increased by saline treatment (Poole et al., 1987). However, Gutkowska et al. (1986) reported that ANP stimulated the basal secretion of vasopressin from pituitaries in vitro but did inhibit KCl-stimulated release of the peptide.

Control o f Cerebrospinal Fluid and Ion Fluxes. Autoradiographic studies have revealed the presence of ANP-binding sites in the cerebral vasculature and in the epithelium of the choroid plexus (Figs. 27G, 29F and G), which suggests that circulating ANP may play a role in the control of cerebrospinal fluid (Bianchi et al., 1986; Gibson et al., 1985; Kurihara et al., 1987b; Quirion et al., 1984). Indeed ANP was found to be present in human cerebrospinal fluid (CSF) (Maruma et al., 1987) and to alter the rate of CSF production (Steardo and Nathanson, 1987). Chabrier et al. (1986, 1987) reported that ANP binds to isolated bovine brain microvessels. The presence of both ANP- and All-binding sites led this group to suggest "that the two systems, which seem to be highly involved in an opposite fashion in the regulation of blood pressure and fluid homeostasis, can also act as mutual antagonists in the control of brain microcirculation and may also have important implications in blood-brain barrier function."

It has been suggested that the presence of ANP-binding sites in the choroid


plexus, ependyma, and linings of ventricles could be related to a possible role of ANP peptides in the control of ion fluxes across the brain cerebrospinal fluid barrier and the production of CSF (Quirion et al., 1984). The presence of ANP-binding sites in the ciliary processes of the retina (Quirion et al., 1984) further suggests that these peptides may be involved with the production of the aqueous humor of the eye.

ANP-Bind ing Sites in White Matter. Autoradiographic studies have further revealed specific binding of ANP to white matter structures such as the corpus callosum (Figs. 27A-29A), the anterior commissure (Figs. 27A-G), and the lateral olfactory tract (Figs. 27A-E) (Gibson et al., 1985; Quirion et al., 1984). In addition, binding sites were observed in the fornix (Figs. 27F-H), the stria medullaris (Figs. 27F-28A), and the internal capsule (Figs. 27G-28G). It is important to note that not all white matter contained an appreciable density of binding sites. However, we have noted what appears to be a considerable "background" of binding which was not observed on sections containing nonspecific binding sites. Although these binding sites could represent receptors en passant to neuronal terminal regions, we suggest an alternative explanation, i.e., the presence of binding sites in astrocytes and/or oligodendroglia which are known to be present in white matter. Astrocytes and oligodendroglia are present in much greater numbers in large myelinated bundles of axons such as the corpus callosum and anterior commissure in comparison with the gray matter (Vaughan, 1984). This suggestion is supported by the demonstration that ANP elevates the concentration of cyclic GMP in astroglia-rich brain-cell cultures (Friedl et al., 1985). Of course, the "background" diffuse binding may also be a reflection of capillary binding of ANP (Chabrier et al., 1987).

Anterior Pituitary and Second Messengers. The localization of binding sites for ANP in various peripheral tissues has been partially confirmed by studies of second messengers (for review see Cantin and Genest, 1985). Waldman et al. (1984) originally reported that in homogenates ANP activates guanylate cyclase and increases the levels of cGMP in several peripheral organs (intestine, lung, liver, adrenal medulla) but not in the brain. However, Takayanagi et al. (1986) reported that ANP stimulated cGMP production in rat brain slice preparations. Several groups have shown that ANP does stimulate cGMP synthesis in the anterior pituitary (Abou-Samra et al., 1987; Heisler et al., 1986; Takayanagi et al., 1986). The anterior pituitary is an organ that demonstrates a large mismatch between ANP receptor binding (+4) and immunoreactivity (nondetectable) (Table I; Fig. 30C) (Kurihara et al., 1987b; yon Schroeder et al., 1985). This suggests the possibility that ANP reacts with the anterior pituitary via the median eminence and/or the blood.

Only few reports are available that investigate a possible coupling between cGMP activation and.anterior pituitary hormonal secretion. Several studies indicate tht there are no demonstrable effects of ANP on hormone secretion, although cGMP is stimulated. In cultured pituitary cells ANP had no effect on ACTH, growth hormone, prolactin, and thyrotropin-stimulating hormone release (Abou-Samra et al., 1987, Heisler et al., 1986). In the superfused rat pituitary-cell system, ANP was not found to influence basal release of prolactin, growth


hormone, and thyrotropin-stimulating hormone (Horvath et al., 1986). However, others have shown that in anterior pituitary cultured ceils, ANP attenuated basal and corticotropin-releasing factor-induced secretion of ACTH (Samson and Eskay, 1986; Shibasaki et al., 1986). ANP has indeed been shown to inhibit basal and corticotropin-releasing factor-stimulated adenylate cyclase activity in anterior pituitary homogenates (Anad-Strivasta et al., 1985). On the other hand, in the superfused rat pituitary, ANP at high concentrations induced a small but significant stimulation of the release of ACTH (Horvath et al., 1986). Addition- ally, ANP stimulated the release of luteinizing hormone (LH) and follicle- stimulating hormone (FSH), while Simard et al. (1986) reported that ANP failed to modify spontaneous or luteinizing hormone-releasing hormone (LHRH)- induced LH secretion. These inconclusive results require further study.

Habenular-Interpreduncular System. The medial habenula and interpeduncular nucleus are regions that contain some of the highest levels of ANP immunoreactivity and binding sites (Figs. 27H, 28A-E, 29A-C; Table I). These two nuclei are integrated by the fasciculus retroflexus, a large nerve trunk that also contains a high density of binding sites (Figs. 28E-H). No function has been attributed to the interpeduncular nucleus other than its designation a s a limbic midbrain structure (Nauta 1958). The interpeduncular nucleus has been shown to contain a remarkable variety of neurochemicals, including acetylcholine, catecho- lamines, serotonin, amino acids, and neuropeptides (see Hamill et al., 1984, 1986). The fasciculus retroflexus (habenular-interpeduncular tract) is a major pathway that contains cholinergic nerves that emanate from the habenula to terminate in the interpeduncular nucleus (Kataoka et al., 1973; Kuhar et al., 1975). The dense ANP binding in the fasciculus retroflexus appears to be to sites that represent receptors en passant from the habenula to the interpeduncular nucleus. It is likely that the ANP-reactive cell bodies in the medial habenular nucleus project (at least partly) to the interpeduncular nucleus. The relationship of the ANP-binding sites to the known neurochemical systems (ACh, substance P) that project from the habenula to the interpeduncular nucleus is unknown and needs to be studied. Knowledge of an ANP neuronal system, along with possible receptor binding sites, constitutes additional pieces of the puzzle that bring us closer to revealing the functions of the habenular-interpeduncular nuclei.

CONCLUSIONS

Studies of the presence of ANP immunoreactivity and receptor binding sites in the central nervous system have revealed unusual sites of interest. As a result, numerous studies have appeared that indicate that brain ANP is implicated in the regulation of blood pressure, fluid and sodium balance, cerebral blood flow, brain microcirculation, blood-brain barrier function, and cerebrospinal fluid production. Alteration of the ANP system in the brain could have important implications in hypertensive disease and disorders of water balance in the central nervous system.


ACKNOWLEDGMENTS

The work was supported P6086M and P56F6. We want assistance.

by Austrian Scientific Research Funds, Grants to thank Ms. Christa Wolfbauer for technical

NOMENCLATURE

a

aa ab abp a c

a c o

al am amb ant ap apm asl BCI c

CA CAI CC CCA ccgm cd CFV CgF2 ChP ci cl CO cod c o v

cp CSDV CT ct cul cv

dcgl DP

Nucleus accumbens Area amygdaloidea anterior Nucleus amygdaloideus basalis Nucleus amygdaloideus basalis posterior Nucleus amygdaloideus centralis Nucleus amygdaloideus corticalis Nucleus amygdaloideus lateralis Nucleus amygdaloideus medialis Nucleus ambiguus Lobus anterior of the pituitary Area postrema Area pretectalis medialis Ansa lenticularis Brachium colliculi inferioris Nucleus caudatus Commissura anterior Capsula interna Crus cerebri Corpus callosum Nucleus centralis corporis geniculati medialis Cornu dorsale Commissura fornicis ventralis Cortex cinguli-cortex frontalis, area 2 Plexus chorioidalis Colliculus inferior Claustrum Chiasma opticum Nucleus cochlearis dorsalis Nucleus cochlearis ventralis Nucleus caudatus putamen Commissura supraoptica dorsalis, pars ventralis (Meynert) Corpus trapezoideum Nucleus corporis trapezoidei Nucleus cuneatus lateralis Cornu ventrale Nucleus dorsalis corporis geniculati lateralis Decussatio pyramidis


DPCS D s c

F FC FD FH FL FLM FMT FMTG FR FS FV g GD

gP HI HIA hi hm ic io ip lc LL lid llr llv LM mcgm ME MFB na n c o

n c s

n c u

ndm nha nhp nic nil nist nistd nistv nlo nml

Decussatio pendunculi cerebellarium superiorum Lamina dissecans cortex entorhinalis Fornix Fasciculus cuneatus Funiculus dorsalis Fimbria hippocampi Funiculus lateralis Fasciculus longitudinalis medialis Fasciculus mamillothalamicus Fasciuculus mamillotegmentalis Fasciculus retroflexus Fornix superior Funiculus ventralis Nucleus gelatinosus thalami Gyrus dentatus Globus pallidus Hippocampus Hippocampus anterior Nucleus habenulae lateralis Nucleus habenulae medialis Nucleus interstitialis (Cajal) Nucleus olivaris inferior Nucleus interpeduncularis Locus coeruleus Lemniscus lateralis Nucleus lemnisci lateralis dorsalis Nucleus lemnisci lateralis rostralis Nucleus lemnisci lateralis ventralis Lemniscus medialis Nucleus marginalis corporis geniculati medialis Eminentia mediana Fasciculus medialis prosencephali (medial forebrain bundle) Nucleus arcuatus Nucleus commissuralis Nucleus centralis superior Nucleus cuneiformis Nucleus dorsomedialis hypothalami Nucleus hypothalamicus anterior Nucleus hypothalamicus posterior Nucleus intercalatus Lobus neurointermedialis of pituitary Nucleus interstitialis striae meduUaris Nucleus interstitialis striae terminalis pars dorsalis Nucleus interstitialis striae terminalis pars ventralis Nucleus linearis oralis Nucleus mamillaris lateralis


n m m

np npd npe npf npmv npV npv nrd nrp n f v

n s c

n s o

ntd ntdl nts ntv ntV ntVd nvm nlII nV nVI nVII nX nXII oap ol ope OS

OVLT P P PCI PCMA PCS pf ph pi po pol pom pos ps pt pv

Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus

mamillaris medialis parabrachialis ventralis parabrachialis dorsalis periventricularis hypothalami perifornicalis premamillaris ventralis sensorius principalis nervi trigemini paraventricularis hypothalami reticularis medullae oblongatae, pars dorsalis reticularis paramedianus reticularis medullae oblongatae, pars ventralis suprachiasmaticus supraopticus tegmenti dorsalis (Gudden) tegmenti dorsalis lateralis tractus solitarii tegmenti ventralis (Gudden) tractus spinalis nervi trigemini tractus spinalis nervi trigemini, pars dorsomedialis ventromedialis hypothalami originis nervi occulomotorii originis nervi trigemini originis nervi abducentis originis nervi facialis originis dorsalis nervi vagi originis nervi hypoglossi olfactorius anterior, pars posterioris tractus olfactorii lateralis preolivaris externus

Nucleus olivaris superior Organum vasculosusm lamina terminalis Tractus corticospinalis Nucleus pretectalis Pedunculus cerebellaris inferior Pedunculus corporis mamillaris Pedunculus cerebellaris superior Nucleus parafscicularis Nucleus prepositus hypoglossi Cortex piriformis Nucleus pontis Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus

preopticus lateralis preopticus medialis preopticus suprachiasmaticus parasolitarius paratenialis periventricularis thalami


r r e

rd rdp r e

rgi rh rl rm r o

rpc rp rpo rpoc rpoo RSA rtp S

SAM sd sdi sdl sf sfo sg SGC SGCd SGCv sgm sgp sgs sgV sl SM s m

s a c

snl snr SO spf ST st SUM s u t

tam tar

Nucleus ruber Area retrochiasmatica Nucleus raphe dorsalis Nucleus raphe dorsalis, pars posterior Nucleus reuniens Nucleus reticularis gigantocellularis Nucleus rhomboideus Nucleus reticularis lateralis Nucleus raphe magnus Nucleus raphe obscurus Nucleus reticularis parvocellularis Nucleus raphe pallidus Nucleus raphe pontis Nucleus reticularis pontis caudalis Nucleus reticularis pontis oralis Cortex retrosplenialis agranularis Nucleus reticularis tegmenti pontis Septum Stratum album mediale colliculi superioris Nucleus dorsalis septi Nucleus dorsalis septi, pars intermedia Nucleus dorsalis septi, pars lateralis Nucleus fimbrialis septi Organum subfornicale Nucleus suprageniculatus facialis Substantia grisea centralis Substantia grisea centralis, pars dorsalis Substantia grisea centralis, pars ventralis Stratum griseum mediale colliculi superioris Stratum griseum profundum colliculi superioris Stratum griseum superficale colliculi superioris Substantia gelatinosa trigemini Nucleus lateralis septi Stria medullaris thalami Nucleus medialis septi Substantia nigra, zona compacta Substantia nigra, zone lateralis Substantia nigra, zona reticularis Stratum opticum colliculi superioris Nucleus subparafascicularis Stria terminalis Nucleus triangularis septi Decussatio supramamillaris Nucleus subthalamicus Nucleus anterior medialis thalami Nucleus anterior ventralis thalami


td tl tip tm tml tmm TO TOL tpm tpo tr TSHT TSTH TSV TT tu tv tvd tvm V vcgl vl v m

vs

vsp zi VII X

Nucleus Nucleus Nucleus Nucleus Nucleus

tractus diagonalis (Broca) lateralis thalami lateralis posterior thalami medialis thalami medialis thalami, pars lateralis

Nucleus medialis thalami, pars medialis Tractus opticus Nucleus tractus optici, pars lateralis Nucleus posteromedianus thalami Nucleus posterior thalami Nucleus reticularis thalami Tractus septohypothalamicus Tractus striohypothalamicus Tractus spinalis nervi trigemini Tractus tectospinalis Tuberculum olfactorium Nucleus ventralis thalami Nucleus ventralis thalami, pars dorsalis Nucleus ventralis medialis thalami, pars magnocellularis Ventriculus Nucleus ventralis corporis geniculati lateralis Nucleus vestibularis lateralis Nucleus vestibularis medialis Nucleus vestibularis superior Nucleus vestibularis spinalis Zona incerta Nervus facialis Nervus vagus

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Atrial natriuretic peptide in the central nervous system of the rat

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