THE JOURNAL OF COMPARATIVE NEUROLOGY 316:287-313 (1992)

Distribution of Substance P-ImmunoreactiveElements in the Preoptic Area and the Hypothalamus of the Rat PHILIP J. LARSEN Institute of Medical Anatomy, Department B, University of Copenhagen, DK-2000 Copenhagen N, Denmark

ABSTRACT The localization and morphology of neurons, processes, and neuronal groups in the rat preoptic area and hypothalamus containing substance P-like immunoreactivity were studied with a highly selective antiserum raised against synthetic substance P. The antiserum was thoroughly characterized by immunoblotting; only substance P was recognized by the antiserum. Absorption of the antiserum with synthetic substance P abolished immunostaining while addition of other hypothalamic neuropeptides had no effect on the immunostaining. The specificity of the observed immunohistochemical staining pattern was further confirmed with a monoclonal substance P antiserum. The distribution of substance P immunoreactive perikarya was investigated in colchicinetreated animals, whereas the distribution of immunoreactive nerve fibers and terminals was described in brains from untreated animals. In colchicine-treated rats, immunoreactive cells were reliably detected throughout the preoptic area and the hypothalamus. In the preoptic region, labeled cells were found in the anteroventral periventricular and the anteroventral preoptic nuclei and the medial and lateral preoptic areas. Within the hypothalamus, immunoreactive cells were found in the suprachiasmatic, paraventricular, supraoptic, ventromedial, dorsomedial, supramammillary, and premammillary nuclei, the retrochiasmatic, medial hypothalamic, and lateral hypothalamic areas, and the tuber cinereum. The immunoreactive cell groups were usually continuous with adjacent cell groups. Because of the highly variable effect of the colchicine treatment, it was not possible to determine the actual number of immunoreactive cells. Mean soma size varied considerably from one cell group to another. Cells in the magnocellular subnuclei of the paraventricular and supraoptic nuclei were among the largest, with a diameter of about 25 km, while cells in the supramammillary and suprachiasmatic nuclei were among the smallest, with a diameter of about 12 p m . Immunoreactive nerve fibers were found in all areas of the preoptic area and the hypothalamus. The morphology, size, density, and number of terminals varied considerably from region to region. Thus, some areas contained single immunoreactive fibers, while others were innervated with such a density that individual nerve fibers were hardly discernible. During the last decade, knowledge about neural organization of rodent hypothalamic areas and mammalian tachykinin biochemistry has increased substantially. In the light of these new insights, the present study gives comprehensive morphological evidence that substance P may be centrally involved in a wide variety of hypothalamic functions. Among these could be sexual behavior, pituitary hormone release, and water homeostasis. Key words: immunohistochemistry,tachykinins, circumventricular organs, paraventricular nucleus,

neuronal connectivity, sexual behavior, water homeostasis

Accepted October 4,1991

O

1992 WILEY-LISS, INC.

P.J. LARSEN Substance P (SP) is probably one of the most widely studied and best characterized neuropeptides in mammalian tissues, including the central nervous system (Nicoll, '80; Jessel, '83; Pernow, '83; Maggio, '88). SP is a member of the tachykinin peptide family, which in mammals additionally includes neurokinin A ( N U ) , neuropeptide K (NpK), and neurokinin B (NkB) (Kangawa et al., '83; Kimura et al., '83; Minamino et al., '84; Kawaguchi et al., '86). SP, NkA, and NpK are encoded by the same gene, preprotachykinin A (PPT-A), which by alternative posttranscriptional splicing gives rise to a-, P-, and y-PPT-A mRNA (Nawa et al., '83, '84; Kawaguchi et al., '86; Krause et al., '87). Most regions of the brain contain both a-PPT-A mRNA, which codes for SP only, and P-PPT-A mRNA,

which codes for SP, N U , and NpK; most regions contain roughly two to three times the amount of a-PPT-A mRNA as P-PPT-A mRNA (Nawa et al., '84). Until now no in situ hybridization histochemical studies have been able to differentiate between the messages of the three PPT-As. Therefore, immunohistochemistry is still the technique of choice when a detailed account of tachykinin transmitter content in various neural systems is sought. SP is present throughout the central nervous system with the hypothalamus being one of the regions displaying the highest content (for review, see Nicoll, '80). High amounts of radioimmunoassayable SP are present in the preoptic, periventricular, paraventricular, ventromedial, and arcuate areas (Brownstein et al., '76; Jessop et al., '90).

Abbreviations ac AD AHA AHC AHP

AP Arc AWO AVPv BST BSTe BSTMPL BSTMPM BSTMPI CP DA DBB DMH DMHC DMHD

EP F f fr Gem GP Gu ic Inf S IML LA LH LM LPO LV MaPO mcht MCLH ME MeA MePD MePO ml ML MM MMn MPA MPN MPNc MPNl MPNm mP MPO MRe mt MTu NC opt ox

anterior commissure anterodorsal preoptic nucleus anterior hypothalamic nucleus, anterior part anterior hypothalamic nucleus, central part anterior hypothalamic nucleus, posterior part anterior pituitary lobe arcuate nucleus anteroventral preoptic nucleus anteroventral periventricular nucleus bed nucleus of stria terminalis encapsulated part posterolateral part of medial division posteromedial part of medial division posterointermediate part of medial division cerebral penduncle dorsal hypothalamic area diagonal band of Broca dorsomedial hypothalamic nucleus central part of DMH diffuse part of DMH entopenduncular nucleus nucleus of fields of Forel fornix fasciculus retroflexus gemini nucleus globus pallidus gustatory thalamic nucleus internal capsule infundibular stalk intermediate pituitary lobe lateroanterior hypothalamic nucleus lateral hypothalamic area lateral mammillary nucleus lateral preoptic area lateral ventricle magnocellular preotic area medial corticohypothalamic tract magnocellular nucleus of the lateral hypothalamic area median eminence medial amygdaloid nucleus, anterior part medial amygdaloid nucleus, posterodorsal part median preoptic nucleus medial lemniscus medial mammillary nucleus, lateral part medial mammillary nucleus, medial part medial mammillary nucleus, median part median preoptic area medial preoptic nucleus central part of MPN lateral part of MPN medial part of MPN mammillary pedunde medial preoptic area mammillary recess of third ventricle mammillothalamic tract medial tuberal nucleus nucleus circularis optic tract optic chiasm

periventricular hypothalamic nucleus perifornical nucleus posterodorsal preoptic nucelus posterior hypothalamic area premammillary nucleus, dorsal part premammillary nucleus, ventral part posterior pituitary lobe parastrial nucleus paratenial thalamic nucleus pars tuberalis of anterior pituitary lobe paraventricular thalamic nucleus, anterior part paraventricular nucleus medial parvicellular PVN dorsal parvicellular PVN periventricular parvicellular PVN anterior parvicellular PVN lateral parvicellular PVN posterior magnocellular PVN anterior magnocellular PVN retrochiasmatic area reuniens nucleus rhomboid thalamic nucleus rostra1 interstitial nucleus of medial longitudinal fasciculus reticular thalamic nucleus suprachiasmatic nucleus superior cerebellar penduncle septohypothalamic nucleus substantia inominata stria medullaris submammillothalamic nucleus substantia nigra, pars compacta substantia nigra, pars reticularis supraoptic nucleus supraoptic decussatio sox subparafascicular thalamic nucleus SPF stria terminalis ST strial part of preoptic area StA subthalamic nucleus STh subincertal nucleus Sub1 supramammillary nucleus SUM subparaventricular area SubPV supramammillary decussatio SUMX tuber cinereum TC terete hypothalamic nucleus Te third ventricle 3v tuberal magnocellular nucleus TM ventromedial thalamic nucleus VM ventromedial hypothalamic nucleus VMH anterior part of VMH VMHA central part of VMH VMHC dorsomedial part of VMH VMHDM posterior part of VMH VMHP ventrolateral part of VMH VMHVL ventral pallidum W ventral posteromedial thalamic nucleus VPM ventral reuniens nucleus VRe zona incerta ZI zona incerta, dorsal part ZID zona incerta, ventral part ZIV

Pe PeF PD PH PMD PMV PP PS PT PTu PVA PVN mpPVN dpPVN pePVN apPVN lpPVN pmPVN amPVN RCh Re Rh RI Rt SCN SCP SHY SI SM SMT SNC SNR SON

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SUBSTANCE P IN THE HYPOTHALAMUS Immunohistochemical studies have revealed that within the preoptic area and the hypothalamus, SP-immunoreactive (SP-IR) cells are present in many nuclei, and various densities of SP-IR fibers are present in nearly all nuclei (Cuello and Kanazawa, '78; Ljungdahl et al., '78; Panula et al., '84). These morphological findings have largely been confirmed by in situ hybridization histochemistry studies (Harlan et al., '89; Warden and Young, '88; Warden, '89). Moreover, autoradiographic binding experiments have shown that tachykinin receptors of the NK-1 subtype, having SP as the endogenous ligand, are present in high numbers in the hypothalamus (Saffroy et al., '88; Mantyh et al., '89). Several lines of evidence indicate that SP influences the anterior pituitary secretion of prolactin, growth hormone, and gonadotropins (for review, see Aronin et al., '86; Jessop et al., '91b). Not much is known about the SPergic innervation of hypothalamic cells producing and releasing hypothalamic factors, but morphological studies indicate that SP could either influence hypothalamic perikarya, neuronal terminals in the external zone of the median eminence, or be released to the portal vascular system (Tsuruo et al., '83, '87, '91; Mikkelsen et al., '89). In addition to actions in the hypothalamo-pituitary axis, SP and related tachykinins have been shown to exert behavioral actions on food and water intake (Fitszimons and Evered, '78; de Car0 et al., '80; Massi et al., '88). SP elicits centrally mediated antidipsogenic and antidiuretic actions and stimulates vasopressin secretion from the posterior pituitary (Haldar et al., '79; de Car0 et al., '80; Chowdrey et al., '90). Furthermore, SP is intimately involved in the hormonal induction of sexual behavior in the rat. Injections of SP into the medial preoptic and anterior hypothalamic areas of male rats significantlyreduce mounting and copulation latencies (Dornan and Malsbury, '87, '89). In the female rat, SP acts as a transmitter inducing sexual receptivity. This action is thought to be mediated via SPergic cell bodies in the ventromedial hypothalamic nucleus projecting to the midbrain central gray (Dornan et al., '90). The hypothalamus plays an important role in the generation and maintenance of most homeostatic behavioral actions, and hypothalamic SP-containing neuronal pathways are likely to influence at least some of these hypothalamic actions. Since the advent of earlier studies describing the distribution of SPergic cell bodies and fibers in the hypothalamus, the knowledge of hypothalamic organization has been considerably extended. In this context no comprehensive study of the detailed topographical distribution of hypothalamic SPergic elements has yet been performed. The present study was designed to assess further the anatomical localization and morphological detail of the SP neurons in the preoptic area and hypothalamus of the rat brain in order to associate morphological heterogenity of SP neuron populations with reported functional sublocalizations. To accomplish this assessment, light microscopic analyses of cytoarchitecture, distribution of perikarya, nerve fibers, and varicosities of immunohistochemically identified SP neurons were used in the brains of colchicine- and noncolchicine-treatedrats. The existence of additional mammalian tachykinins with a high degree of similarity in amino acid sequences was unknown when the first accounts on SPergic systems were given. Since this phenomenon often results in problems with cross-reactivity of antisera, it raises the possibility that earlier studies have unknowingly

confused SP with other tachykinins. In the present study, two different antisera were used for immunohistochemistry, and one of these is characterized in great detail with dot blots and preabsorptions with a large number of hypothalamic neuropeptides.

MATERIALS AND METHODS Antisera Rabbit antiserum (code no. 250-2) raised against SP was generously provided by J.J. Holst (Copenhagen, Denmark). Rat monoclonal anti-SP antiserum was purchased from Sera-Lab (Crawley Down, Sussex, U.K.). Biotinylated swine anti-rabbit IgG was obtained from Dakopatts (Copenhagen, Denmark), and biotinylated rabbit anti-rat serum was obtained from Vector Laboratory (Burlingame, CA).

Immunohistochemistry Fifteen male Wistar rats weighing 200 g and kept under standard laboratory conditions were used for the immunohistochemical studies. Twenty-four hours prior to fixation eight animals received an injection of colchicine (120 pg in 10 pl 0.9% NaC1) into the third ventricle. Prior to fixation animals were anesthetized with tribromethanol (20 mgl 100 g body weight). Then they were perfused transcardially with potassium phosphate-buffered saline (KPBS) containing heparin (15,000 IU/1) followed by 300 ml4% paraformaldehyde dissolved in 0.1 M phosphate buffer. The brains were rapidly removed from the skull and postfixed in the same fixative for 4-8 hours. The brains were cryoprotected in a 30% sucrose-KPBS solution for 2 days. One-in-six series of 40 pm thick frontal sections were cut in a cryostat and collected in KPBS. Sections to be tested with anti-SP were preabsorbed in 10% swine serum for 30 minutes. Thereafter, the sections were incubated at 4°C for 24 hours in rabbit anti-SP antiserum diluted 1:1,000 in KPBS containing 1%bovine serum albumin (BSA) and 0.3% Triton-X-100 (KPBS-TI. Another group of sections was incubated under identicalconditionsin a monoclonal anti-SP antiserum diluted 1:1,000. Prior to incubation in the secondary antiserum, the sections were rinsed for 3 x 10 minutes in KPBS-T. Then the sections were incubated in either biotinylated swine anti-rabbit serum or biotinylated rabbit anti-rat serum diluted 1:400 in KPBS-T for 1hour at room temperature, and washed again. Thereafter, the sections were incubated in peroxidase-conjugatedstreptavidin (#K377, Dakopatts) diluted in KPBS-T for 1 hour. After the sections were washed first for 10 minutes in KPBS-T, second for 10 minutes in KPBS, and finally for 10 minutes in Tris-buffered saline (pH 7.6) they were incubated with diaminobenzidine (DAB; 25 mg in 100 ml Tris-buffered saline) containing 35 ml35% H,O, for 10-30 minutes. After a rinse in Tris-buffered saline and then in distilled water the sections were mounted. A series of processed sections were counterstained with thionine. In addition to omitting the primary antiserum and incubating the sections with nonimmune serum, the following controls were carried out.

Immunoblotting model system The polyclonal rabbit anti-SP antiserum was tested for binding to various peptides by employing a previously described method (Larsen et al., '89a). Briefly, the peptides tested for binding to the SP antiserum were dissolved at

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P.J. LARSEN

rising concentrations in redistilled water. The peptides used were alytesin (0.13 and 13 pM); arginine-vasopressin (0.92, 9.2, and 92.0 FM); bombesin (0.12, 1.2, and 12 pM); corticotropin-releasing factor (CRF) (0.21, 2.1, and 21.0 pM); gastrin-releasingpeptide (GRP)'4-27(0.06,0.6,6.0, and 60 pM); ( G ~ U ~ ~ - P ~ ~ ~ ~ (0.06 ) - G Rand P ~6~ pM); - ~ ' Metenkephalin (0.87, 8.7, and 87 FM); neurokinin A (0.88, 8.8, and 88.0 pM); neurokinin B (0.82, 8.2, and 82.0 pM); oxytocin (0.99, 9.9, and 99.0 pM); peptide histidine isoleucine (PHI) (0.17, 1.7, and 17 pM); ranatensin (0.39,3.9, and 39 pM); substance P (0.74, 7.4, and 74.0 pM); substance P sulfoxide (0.37, 3.7, and 37 pM); substance PSn tripeptide (1.5, 15, and 150 pM);and vasoactive intestinal peptide (VIP) (0.15, 1.5, and 15 FM). The peptides were placed on strips of nitrocellulose filter paper in a volume of 5 pl per spot. Following drying of the spots, the filter strips were fixed in paraformaldehyde vapor for 90 minutes, whereafter the filters went through the same immunohistochemical procedure as described for the brain sections.

Absorptions The polyclonal rabbit antiserum was preabsorbed at room temperature for 1hour before use with the following peptides: neurokinin A (8.8and 88.0 pM); substance P (7.4, 37, and 74 pM); substance P9-l1 tripeptide (157 and 314 FM); arginine-vasopressin (0.92,9.2, and 92 pM); oxytocin (0.99,9.9, and 99.0 FM) prior to incubation of the sections.

RESULTS The immunoblotting and preabsorption control experiments revealed that the polyclonal anti-SP serum only recognized SP. When the monoclonal SP antiserum was used, a staining pattern identical to that obtained with the polyclonal antiserum was observed. Colchicine treatment was necessary for the visualization of hypothalamic SP-immunoreactive cell bodies. Although the density of immunoreactive fibers generally was lower in these animals, the overall distribution of stained fibers was not noticably affected by colchicine treatment. However, the distribution of SP-IR cells and fibers reported below is based on results obtained in colchicine-treated and -nontreated rats, respectively. The colchicine injections were given intraventricularly to obtain as wide a spread as possible. However, many regions of the brain may not be reached via this route of administration. Injections in the lateral hypothalamic area did not give rise to a larger number of immunoreactive cell bodies in the hypothalamus, whereas immunoreactive cell bodies not seen after intraventricular injections were observed in the amygdala and the caudate putamen. Animals in which colchicine was accidently infused intraparenchymallywere discarded from the study. The cytoarchitectonic criteria used for the parcellation of the preoptic area and the hypothalamus proper are discussed in Bleier et al., '79. The nomenclature used is largely adopted from Paxinos and Watson ('86).

Soma size and morphologies There was a great disparity in the size of SP-IR somata. Some were large, for instance in the supraopticand paraventricular magnocellular subnuclei (Fig. lc), while others were very small, particularly in the suprachiasmatic nucleus (Fig. lb). Most often the cells were medium sized, i.e., 10-20 pm, and cell bodies of this size were seen in a great number of hypothalamic nuclei. Although it is difficult to exclude with certainty that the morphology of the dendritic

arbor has changed during colchicine treatment, some general comments can be given on cell morphologies. The most predominant SP-IR cell type observed in hypothalamic nuclei was bipolar and of intermediate size (Fig. la). In contrast, in the ventrolateral part of the ventromedial hypothalamic nucleus and the premammillary nuclei the predominant SP-IR cells were multipolar and of intermediate size (Fig. ld,e). Within the latter nuclei, it was often possible to follow part of the dendritic arbor into the neuropil surrounding the nuclei. However, in most other areas it was virtually impossible to get an impression of the extent of the dendritic tree. All regions of the preoptic area and hypothalamus contained immunoreactive s o n s . Generally, the SP-IR fibers were finely calibered and endowed with small varicose boutons en passage.

Preoptic region SP-IR fibers. All parts of the preoptic region contained SP-IR fibers, but distinct regional differences in fiber densities were observed. Within the medial preoptic nucleus (MPN), a clear shift in the fiber density of individual subnuclei was observed in the rostrocuadal extent of the nucleus (Fig. 2A-C). Within the medial part of the MPN (MPNm), the highest density of SP-IR fibers was observed in the rostral part (Fig. 4) with a gradual decrease in caudal direction. Most of the SP-IR fibers reached the MPNm from projections coursing along the periventricular strata of the third ventricle. Few SP-IR fibers were present within the central part of the MPN (Fig. 2B). In contrast to the medial part of the MPN, the density of SP-IR fibers within the lateral part of MPN increased in the caudal direction. In the medial preoptic area (MPO) lying laterally to the MPN1, a moderate density of SP-IR fibers was observed. In the rostral MPO, a high density of fibers was seen in the preoptic suprachiasmatic nucleus and the anteroventral periventricular nucleus (AVPv) (Fig. 6), whereas a moderate to low density of SP-IR fibers was observed in the ventrolateral part of the MPO. At the rostral border of the medial preoptic area, a few immunoreactive fibers coursed from the periventricular hypothalamic nucleus into the organum vasculosum of the lamina terminalis. Some SP-IR fibers entered the preoptic suprachiasmatic nucleus and the AVPv from caudally via the periventricular strata of the third ventricle. Within the rostral tip of the anteroventral preoptic nucleus (AVPO), a moderate number of SP-IR fibers was observed, whereas the caudal part was sparsely innervated. Within the dorsomedial (strial) and central part of the MPO a moderate number of stained fibers was widely dispersed. The MePO and anterodorsal preoptic nucleus were almost devoid of SP-IR fibers. However, further caudally a high number of SP-IR fibers and terminals was seen in the posterodorsal preoptic nucleus as well as in the caudal subnucleus of the parastrial nucleus (PS)(Fig. 2B,C) (cf. Simerly et al., '84 for parcellation). Most of the SP-IR fibers in the PS appeared to enter this nucleus laterally from the medial forebrain bundle. Within the rostral part of the lateral preoptic area (LPO), a moderate number of SP-IR fibers was encountered in the ventromedial half. However, within the dorsolateral part of the LPO corresponding to the medial forebrain bundle (mfb), a high density of varicose fibers was seen. From the mfb, immunoreactive fibers coursed medially along the ventrolateral aspect of the bed nucleus of the stria terminalis into the MPO. SP-IR cell bodies. Numerous intensely stained SP-IR cell bodies were widely dispersed throughout the medial

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SUBSTANCE P IN THE HYPOTHALAMUS \

?Y

e

50 ym Fig. 1. Camera lucida drawing of cell bodies and proximal dendrites and axons from various hypothalamic nuclei. a: Two parvicellular bipolar neurons from the apPVN. b A small neuron from the SCN. c : Two magnocellular neurons from the SON; an axon possessing boutons

en passant is seen leaving one of them. d: A medium-sized neuron from the ventrolateral part of the VMH. e: A group of medium-sized neurons from the SUM. From the latter, axons are clearly seen to leave the somata from two cells. Scale bar = 50 p,m.

preoptic area (Figs. 7, 9). The density of these cells was particularly high within the ventrolateral aspect of the MPO (Fig. 8 ) . The AVPv contained a moderate number of labeled cells, whereas few SP-IR cell bodies were observed in the periventricular hypothalamic nucleus and the suprachiasmatic preoptic nucleus. In the MPNm, few immunoreactive cell bodies were encountered in the ventral part (Figs. 2A-C, 8).Thus, the MPN proper was almost devoid of SP-IR cell bodies whereas the surrounding MPO contained a high number of stained perikarya. In the rostral AWO, a moderate number of SP-IR cell bodies was observed. In the caudal aspect of the MPO, the SP-IR cell population merged with a group of morphologically similar immunoreactive cell bodies in the LPO (Figs. 9, 10).

Medial part of the anterior region of the hypotha1amus

between the anterior hypothalamic area and the periventricular hypothalamus on the one hand and the suprachiasmatic nucleus and the PVN on the other. Within this area, which some authors designate the subparaventricular zone (SubPV) (Watts et al., '87),a dense plexus of coarse SP-IR fibers possessing a high number of varicosities was observed (Fig. 3A-C). In the part of the SubPV lying dorsomedially to the central part of the AH, the fiber orientation was predominantly parallel to the demarcation of the AH. Medial to the AH, the immunoreactive fibers of the SubPV merged with a dense fiber plexus in the periventricular hypothalamic nucleus (Pel. Some of the fibers in the SubPV entered this zone from the caudal part of the suprachiasmatic nucleus. However, the largest contingent of stained fibers in the SubPV appeared to enter this area from the rostrolaterally situated bed nucleus of the stria terminalis (BST) (Figs. 3,11-13). From the dorsomedial aspect of the

SP-IR fibers. In the present study, the anterior hypothalamic nucleus (AH) is defined as a loose collection of heterogenous cells continuous rostrally with the MPN and extending to the caudal margin of the paraventricular hypothalamic nucleus (PVN). Within the AH a low density of uniformly distributed SP-IR fibers and terminals was observed (Fig. 3A-C). AH is surrounded dorsomediallyby a comma-shaped cell-sparse zone, or capsule, intervening

Figs. 2-5 (pages 292-293). Series of camera lucida drawings from the preoptic area and the hypothalamus. The positions of immunoreactive cells and fibers on two 40 pm sections combined into one drawing are shown in order from rostral (Fig.2A) to caudal (Fig. 5D).Each dot represents one to five cells; the numbers of fibers are not the actual numbers but show the relative density between various areas.

rnpPVN

IoPVN

Figures 2 and 3

ME

Figures 4 and 5

VMHVL

\

4

Figures 6-10

SUBSTANCE P IN THE HYPOTHALAMUS

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Figs. 11-13. Fig. 11: Low powerphotomicrogmph from the midportion of t h e BST. A high density of SP-IR terminals and fibers is seen in the encapsulated part of the BST. Ventrally, in the BSTMPI scattered SP-IR somata are seen. Apparently SP-IR nerve fibers enter the BST from t h e stria terminalis. Scale bar = 200 pm. Figs. 12, 13:Low power

photomicrographs of two sections from t h e caudal BST. Figure 12 is 200 +m caudal to Figure 11. A dense SP-IR projection is seen to enter the medial hypothalamic region from t h e BST just ventral to the apPVN (open curved arrow). Scale bars = 200 pm.

SubPV a moderate number of SP-IR fibers coursed into the parvicellular parts of the PVN. Ventrolaterally to the AH, the density of immunoreactive fibers increased substan-

tially when this nucleus was followed from the rostral to the caudal part (Fig. 3C,D). The lateroanterior hypothalamic nucleus (LA) was sparsely innervated by a uniformly distributed plexus of SP-IR fibers (Fig. 3A). Within the suprachiasmatic nucleus (SCN), a moderate density of s p - 1 fibers ~ was observed at the midlevel and caudal pole Of the nucleusr whereas the density Of immunereactive fibers was low in the rostral part of the SCN. At the midlevel of the SCN, it was evident that immunoreactive fibers and terminals were Dredominantly localized in the ventrolateral quadrant, Within the intervening between the two SCN a fiber Of' moderate was Observed (Fig. 23). This fiber plexus was continuous with the high density fiber plexus seen in the retrochiasmatic area and the Pe, from where a few fibers could be followed further dorsally into the SubPV. The PVN, which in the anterior hypothalamic region is the most complex can be subdivided into eight distinct subnuclei (Swanson and Kuypers, ' 8 0 ) . SPergic fibers and terminals were predominantly observed in the

Figs. 6-10. Fig. 6: LOW power photomicrograph from the rostral medial part of the preoptic region. A dense innervation of SP-IR terminals and fibers is seen in the A W v (open curved arrows). Scale Fig. 7:L~~ power photomicrograph from the medial bar = 200 preoptic area. Scattered immunoreactive somata (solid arrows) a r e seen in the medial preuptic area outside the medially situated MPN (open curved arrows). Scale bar = 200 w r n . Fig. 8: Low power photomicrograph from t h e caudal preoptic area. Small solid arrows indicate scattered SP-IR somata in the medial preoptic area. Broken line indicates t h e vcntral demarcation of the MPN, which contains very few immunoreactive somata. Scale bar = 200 pm. Fig. 9: LOW powkr photomicrograph from the border between t h e caudal preoptic region and the rostral hypothalamus. A high number of SP-IR somata (arrows) are seen in the lateral a s well as medial preoptic area. Scale bar = 200 pm. Fig. 10: High power photomicrograph of a medium-sized SP-IK. cell from t h e medial preoptic area. A dcndritic process leaving t h e perikarya is indicated with open arrows. An axon possessing boutons en passant is seen in the lower right (solid arrows). Scale bar = 50 pm.

area

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parvicellular division of the nucleus (Fig. 20). A moderate to high density of stained fibers was observed subependymally in the full rostrocaudal extent of the periventricular parvicellular subnucleus (pePVN). Fibers in the pePVN were rostrally and caudally continuous with SP-IR fibers in the periventricular strata of the third ventricle. In the rostrally situated anterior parvicellular part of the PVN (apPVN), a moderate density of fibers continuous ventrally with the fiber plexus in the SubPV and dorsally with stained fibers in the medial corticohypothalamictract and the paraventricular thalamic nucleus was observed (Fig. 2C). The anterior and medial magnocellular parts of the PVN, which primarily are oxytocinergic, contained a low to moderate innervation of SP-IR fibers. In the intermediate part of the PVN, a moderate to high density of stained fibers was seen in the medial parvicellular subnucleus (mpPVN) (Fig. 20). Different innervation patterns were observed in the ventral (brain stem projecting)and dorsal (median eminenceprojecting) divisions of the mpPVN, with the former having the highest density of SP-IR fibers. The ventral part of the mpPVN receives stained fibers from the ventrally located SubPV, while the dorsal part of the mpPVN receives a contingent of SP-IR fibers from the medially situated pePVN. The posterior magnocellular subnucleus (pmPVN) contained a low to moderate number of stained fibers in the dorsolateral and central part (primarily vasopressinergic) as well as the anteromedial and circumferential part (primarily oxytocinergic) (Fig. 18).In the perimeter surrounding the dorsal, lateral, and ventral aspect of the mpPVN, a dense plexus of SP-IR fibers and terminals and fibers was present (Fig. 20). Occasionally, fibers penetrated into the mpPVN from this neighboring plexus. The dorsal and lateral parvicellular PVN (dpPVN and 1pPVN) contained a moderate to high density of labeled fibers. However, the cell-poor area intervening between the PVN ventrally and the zona incerta dorsally contained a very high density of stained fibers which was continuous with the fibers observed in the dpPVN and lpPVN (Fig. 3D).This high density SP-IR fiber plexus could be followed laterally and dorsally to the fornix and further into the lateral hypothalamic area. Within the supraoptic nucleus (SON)a moderate number of SP-IR fibers was present in the full rostrocaudal extent of the nucleus with highest concentration in the ventral glial lamina. In the perinuclear zone, a moderate density of fibers was observed (Fig. 21). Thus, SP-IR fibers constituted a perinuclear shell surrounding the dorsolateral aspect of the nucleus. Fibers from this position coursed dorsomedially via the tuber cinereum in direction of the retrochiasmatic area and the median eminence. SP-IR cell bodies. In the most rostral part of the anterior hypothalamic region, a moderate number of multiform, medium-sized SP-IR neurons was observed surrounding the fornix (Fig. 3A). These cells were likely to be positioned in the most caudal aspect of the BST. However, in the vaguely defined perifornical area, a moderate number of SP-IR cell bodies with a similar morphologywas observed throughout the rostrocaudal extent of the hypothalamus (Fig. 31). Occasionally medium-sized, fusiform, perikarya were encountered in the AH area proper. Within the SCN, a moderate number of immunoreactive perikarya showinga clearly regional distribution was present (Fig. 23). The number of immunoreactive perikarya increased when the nucleus was followed from rostral to caudal, decreasing again in the extreme caudal part of the

P.J. LARSEN SCN. The intermediate part contained a moderate number of small, ovoid (10-11 pm) perikarya (Figs. 23, 24). These perikarya were positioned medially in the ventrolateral quadrant along the obliquely orientated border to the dorsomedial part of the SCN (vasopressinergiccell group). Within the poorly defined area sometime referred to as the lateroanterior hypothalamic area, a moderate number of SP-IR perikarya was seen. This area is situated between the SCN and the supraoptic nucleus on the one hand and the lateral anterior hypothalamic nucleus and the optic tract on the other, but the group of SP-IR cell bodies appeared to be continuous with the rather diffusely distributed SP-IR cell group in the lateral preoptic and lateral hypothalamic areas (Fig. 9). Immunoreactive cell bodies were observed in all subnuclei of the PVN. The most rostral part of the apPVN contained very few immunoreactive cell bodies, whereas in the more caudal aspect of the apPVN a moderate number of medium-sized,bipolar SP-IR perikarya was observed (Figs. 14, 15). Within the amPVN and mmPVN immunoreactive perikarya were rarely observed. However, those found had a morphology similar to that described later for the pmPVN. Throughout the rostrocaudal extent of the pePVN a low number of SP-IR perikarya was seen. The immunoreactive perikarya observed in the pePVN were either small, ovoid, or medium-sized and bipolar, with an overall orientation parallel to the ventricular wall (Fig. 16). These cell bodies were continuous with the SP-IR cell group observed in the surrounding periventricular hypothalamic nucleus. The mpPVN contained the highest number of SP-IR cell bodies observed in the PVN complex (Figs. 17, 20). The perikarya observed in the mpPVN were predominantly bipolar and medium-sized. Dorsally, in the dpPVN a small number of medium-sized cell bodies was observed. In contrast, the lpPVN contained a high number of mediumsized, bipolar perikarya (Figs. 3C,D, 4A). Within the pmPVN a moderate number of large, multiform SP-IR perikarya was observed (Figs. lc, 18, 19). The stained cell bodies in the pmPVN were restricted to the central and dorsolateral part of the subnucleus, corresponding to the part where vasopressinergiccells predominate. Throughout the rostrocaudal extent of the SON, large immunoreactive cell bodies were present (Figs. lc, 21, 22). However, the number of stained perikarya increased in the caudal direction. Within the nucleus, the stained perikarya

Figs. 14-19. Fig. 14 Low power photomicrograph showing the rostral apPVN. A moderate number of SP-IR somata is seen within the borders of the nucleus (solid arrows). Scale bar = 200 pm. Fig. 15: Medium power photomicrograph from the caudal apPVN. Mediumsized multiform SP-IR cell bodies are indicated with solid arrows. Scale bar = 100 wm. Fig. 16:High power photomicrograph from the pePVN. The predominant cell type seen in this area is small and ovoid (solid arrows) and some of the immunoreactive cells are located very close to the ependyma (open arrow). Scale bar = 50 pm). Fig. 17: Medium power photomicrograph from the mpPVN. Immunoreactive cell bodies are either medium-sized and multiform (open arrows) or small and ovoid (solid arrows). Scale bar = 100 pm. Fig. 18: Low power photomicrograph of the PVN complex. The pmPVN is circumscribed by a broken line and within the border of this subnucleus a moderate number of large SP-IR somata is seen (slender solid arrows).Within the mpPVN a moderate number of medium-sized SP-IR somata is seen (bold solid arrows). Scale bar = 200 pm. Fig. 19: High power photomicrograph from the pmPVN of Figure 18.A population of large SP-IR perikarya with no apparent cellular extensions is seen. Scale bar = 50 pm.

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Figures 14-19

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200 pm

Fig. 20. Camera lucida drawing from the midportion of the PVN complex showing immunoreactive cell bodies and nerve fibers. A clear difference in the density of innervation is seen between the ventral

(brain stem projecting) and the dorsal (median eminence projecting) part of the mpPVN. A low density of SP-IR fibers is present in the pmPVN.

were almost exclusively localized in the ventral part corresponding to the area preferentially housing vasopressinergic cells.

perinuclear shell of the VMH, a high density of fine varicose immunoreactive fibers was present (Figs. 3C,D, U-C, 35). This fiber plexus was laterally continuous with fibers in the mfi and rostrally with fibers in the lateroanterior hypothalamic area. In the VMH proper, SP-IR fibers accumulated in the ventrolateral part (Figs. 33, 34). However, when moving cauddly the number of SP-IR fibers increased in the dorsolateral part of the perinuclear shell (Fig. 4A-C). From this area, immunoreactive fibers could be followed to the dorsomedial hypothalamic nucleus (DMH) and the perifornical area. The arcuate nucleus (Arc) contained a high density of immunoreactive fibers throughout its rostrocaudal extent. Consideringthe high density of SP-IR fibers within the Arc, remarkably few of the varicose fibers could be followed into the medially situated median eminence (Fig. 37). In contrast, a considerable number of stained fibers could be followed dorsally into the periventricular hypothalamic nucleus and laterally into the TC. However, the direction of projecting fibers was in this instance evaluated from colchicine-treated animals because of a rather peculiar pattern of SP-IR elements observed in nontreated animals. As demonstrated in Figure 26, the SP-IR material was densely and diffusely distributed throughout the neuropil in the lateral zone of the median eminence (ME) bordering on the Arc. This area closely corresponds to that defined as the lateral

Region of the tuber cinereum SP-IR fibers. Within the area of the tuber cinereum (TO, which is a loose collection of polymorphic cells lying around and between well-defined nuclear areas in the middle part of the hypothalamus, a substantial number of immunoreactive nerve fibers was observed. A moderate to high density of SP-IR fibers was observed in the supraoptic decussatio (sox) (Figs. 3B-D, 30). In the lateral aspect of the sox, the SP-IR fibers were preferentially orientated perpendicularly to the ventrally situated optic tract, whereas in the medial part of the sox the general orientation of the stained fibers was parallel to the ventral surface of the hypothalamus. In the retrochiasmatic area (RCh),a high density of fine, varicose SP-IR fibers was seen in the midline portion (Fig. 3A,B). From sagittal sections it was obvious that some SP-IR fibers coursed caudally from the RCh to enter the rostral aspect of the median eminence. Within the part of the TC lying laterally to the ventromedial hypothalamic nucleus (VMH), a moderate number of SP-IR fibers was observed (Fig. 3B). Throughout the rostrocaudal extent of the perifornical area, a high density of stained fibers was observed. Within the ventrolateral

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Figs. 21-24. Fig. 21: Low power photomicrograph showing SP-IR perikarya in t h e SON. The immunoreactive perikarya a r e preferentially located in the ventral part of t h e nucleus. Scale bar = 100 pm. Fig. 22: High power photomicrograph of the section shown in Figure 21. Large immunoreactive perikarya a r e seen in t h e ventral part of t h e SON. Scale bar = 50 pm. Fig. 23: Low power photomicrograph from the midportion of the SCN. A high density of SP-IR nerve fibers and

terminals is seen in t h e ventrolateral part of the nucleus (solid curved arrows). Within the nucleus a moderate number of immunoreactive cells (small solid arrows) is seen in the zone bordering the ventromedial part of t h e nucleus (open curved arrow). Scale bar = 200 pm. Fig. 24: High power photomicrograph o f t h e section from Figure 23 showing the small, ovoid SP-IR perikarya in the SCN. Scale bar = 50 pm.

border zone of the neurohemal region of the ME by Krisch and Leonhardt ('78). Examination of sagittal sections revealed a moderate density of SP-IR fibers and terminals in the internal and external zone of the ME (Fig. 25). Within the internal zone,

stained fibers could be followed rostrocaudally in the direction of the infundibular stalk (Fig. 27). Within the external zone of the ME, SP-IR terminals were seen in the palisade layer surrounding the portal capillaries (Fig. 28). However, the immunoreactive terminals were not frequently seen in

Figures 25-29

SUBSTANCE P IN THE HYPOTHALAMUS direct apposition to the capillary lumen. Within the pial tissue covering the pars tuberalis of the anterior pituitary lobe, several longitudinally orientated SP-IR fibers were seen (Fig. 28). Occasionally, these fibers entered the pars tuberalis, but they were never observed to enter the external zone of the ME. SP-IR fibers were observed to enter the posterior pituitary lobe from the infundibular stalk. Within the posterior pituitary lobe, a high density of stained fibers with varying morphology was seen. Most predominant were large, varicose terminals in apposition to the vascular lumen of blood vessels (Fig. 29). In addition, a small number of varicose fibers and smaller terminals with no apparent contact to blood vessels was seen. In the diffuse part of the dorsomedial hypothalamic nucleus (DMH), a moderate to high density of SP-IR fibers was observed. Dorsally, the immunoreactive fibers were continuous with a plexus of SP-IR fibers seen in the dorsal hypothalamic area (Fig. 4B-D). In the midportion of the DMH, the compact part was clearly seen to contain a much lower density of stained fibers than the surrounding diffuse part (Fig. 38). A very high density of stained fibers was seen in the ventrolateral zone constituting a border between the central and diffuse part (Figs. 38, 39). Caudally, where the DMH merges with the arcuate nucleus, identical densities of SP-IR fibers and terminals were observed within the two nuclei (Fig. 5B). SP-ZR eel1 bodies. A moderate number of mediumsized, multiform SP-IR cells was observed in the RCh (Figs. 30, 31). Laterally, this cell group was continuous with a larger population of morphologically similar cells in the ventral part of the TC. The cell bodies within the TC were loosely arranged and rostrally continuous with the SP-IR cell population in the lateroanterior hypothalamic area. Laterally, the SP-IR cells of the TC merged with the cell group localized in the lateral hypothalamic area. The highest number of SP-IR cell bodies was observed in the VMH (Figs. 33-35). Within the VMH, the stained cells were almost exclusively confined to the ventrolateral part of the nucleus. The SP-IR cell bodies were medium-sized and multipolar (Figs. Id, 36), and frequently dendritic arbors were seen to penetrate into the ventrolaterally situated perinuclear shell (Fig. 34). Within the rostral part of the diffuse DMH, a moderate number of medium-sized, multiform SP-IR cell bodies was observed (Fig. 41). However, when followed caudally the

Figs. 25-29. Fig. 25: Low power photomicrograph of a sagittal section of the ventral part of the third ventricle with the adjacent pituitary gland. A high density of SP-IR elements is seen in the caudal median eminence, the infundibular stalk, and the posterior pituitary lobe. Scale bar = 200 Fm. Fig. 2 6 Low power photomicrograph of the median eminence from a non-colchicine-treated animal. A high density of SP-IR material is seen within the borders of the neurohemal region of the ME (solid arrows). Scale bar = 200 pm. Fig. 2 7 High power photomicrograph from a sagittally cut section showing the internal zone of the ME. A longitudinally orientated SP-IR nerve fiber possessing boutons en passant is indicated with solid arrows. Scale bar = 50 pm. Fig. 28: High power photomicrograph from a sagittally cut section showing the external zone of the ME and the adjacent PTu. A relatively high density of SP-IR terminals is seen in the palisade layer surrounding the portal capillaries ( c ) . Within the pial tissue covering the PTu longitudinally orientated immunoreactive fibers are seen (solid arrows). Scale bar = 50 pm. Fig. 29: High power photomicrograph from the posterior pituitary lobe showing an SP-IR nerve fiber possessing large peptidergic terminals (solid arrows). Scale bar = 20 pm.

301 number of immunoreactive cell bodies increased markedly (Fig. 40). At the midlevel of the DMH, where the wing-like central part is well defined, the SP-IR cell bodies were clearly seen to be confined to the surrounding diffuse part (Fig. 39). A moderate number of rather large SP-IR cells in the DMH was continuous laterally with a scattered group of immunoreactive cells in the PeF. Throughout the rostrocaudal extent of the PeF, a moderate number of these rather large SP-IR cell bodies was observed (Fig. 32). Caudally, as the DMH merges with the Arc, the SP-IR cell population of the DMH becomes continuous with that of the Arc. A substantial number of small, ovoid SP-IR cell bodies was observed in the Arc (Fig. 37).A clear regional difference was observed as the number of immunoreactive cell bodies increased when the Arc was followed in the rostrocaudal direction (Figs. 3C,D, 4, 5). Thus, at the level of the caudal termination of the Arc the number of SP-IR cells was high, while virtually none were seen in the rostral part of the nucleus.

Lateral hypothalamic area SP-ZR fibers. The lateral hypothalamic area (LH),which is continuous rostrally with the lateral preoptic area and caudally with the mesencephalic tegmentum, is medially discerned from the medial hypothalamus by an arbitrary borderline corresponding to the sagittal plane through which the fornix descends. Within this area, a moderate density of SP-IR fibers and terminals was observed (Figs. 3, 4). At the midlevel of the PVN, a characteristic high density fiber plexus was observed dorsolaterally to the fornix. Apparently, the fibers entered this position from the dorsolateral wing of the PVN (Fig. 3C,D). Lateral to the fornix, the majority of the SP-IR fibers coursed ventrally, exhibiting a gradual decrease in fiber density. At the caudal pole of the PVN, an additional high density fiber plexus was observed in the dorsal part of the LH. Instead of coursing ventrally, this fiber plexus progressed along the ventral margin of the zona incerta, traversing the mfb to a position dorsal to the entopenduncular nucleus (Figs. 3D, 4A,B). Caudal to the PVN, the density of stained fibers and terminals in the LH gradually decreased. In the caudal LH, a moderate density of SP-IR fibers and many terminals were observed in the lateral hypothalamic magnocellular nucleus (MCLH) (Fig. 4C,D). Another welldefined nucleus in this area, the medial tuberal nucleus (MTu), contained a moderate density of stained fibers and terminals (Figs. 4C,D, 5A). SP-ZR cell bodies. In general, the LH housed a moderate number of medium-sized, multiform cell bodies scattered diffusely throughout the area. However, in the part of the LH lying dorsal to the SON a substantial number of SP-IR perikarya continuous with the diffusely distributed cell group in the lateroanterior hypothalamic area was observed. Caudally, SP-IR cells were preferentially located within the MTu. A small number of faintly stained cell bodies was observed in MCLH.

Mammillary region SP-ZR fibers. The posterior hypothalamic area (PHI, which is rostrally continuous with the dorsal hypothalamic area (DA), contained a moderate density of SP-IR fibers. The majority of the stained fibers could be followed from the PH into the periventricular fiber system of the third ventricle (Fig. 5C,D).

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Figs. 30-33. Fig. 30: Medium power photomicrograph from the retrochiasmatic area. Scattered immunoreactive cells are seen in the region, and ventrally in the supraoptic decussation a high density of SP-IR fibers and terminals is seen. Scale bar = 100 pm. Fig. 31: High power photomicrograph showing medium-sized, multiform SP-IR neur o w of the RCh. An immunorcactive cell (open curved arrow) with clearly recognizcd dendritic processes (solid arrows) is seen. Scale bar =

50 km. Fig. 32: Medium power photomicrograph from the perifornical nucleus. Medium-sized SP-IR somata is seen in the immediate vicinity of t h e fornix (solid arrows). Scale bar = 100 km. Fig. 33: Low power photomicrograph showing the midportion of the VMH. SP-IR somata are preferentially located in the ventrolateral part of the VMH, while the dorsomedial part appears to be dcvoid of immunoreactive elements (open curved arrow). Scale bar = 200 pm.

Within the mammillary region, the highest density of SP-IR fibers was seen in the ventral premammillary nucleus (PMV) (Figs. 42,44). From here, a large contingent of fibers could be followed dorsomedially traversing the dorsal premammillary nucleus (PMD) and further dorsally into the PH (Fig. 42). A smaller, but still substantial, number of

immunoreactive fibers coursed ventrolaterally from the PMV into the lateral hypothalamic area. Within the rostra1 part of the PMD, a moderate to high density of stained fibers traversed the nucleus in its dorsoventral axis. However, further caudally where the PMD merges with the magnocellular neurons of the tuberal

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200 pm

Fig. 34. Camera lucida drawing showing the midportion of the ventromedial hypothalamic nucleus. Medium-sized, multiform SP-IR perikarya are seen in the ventrolateral portion of the nucleus, while the

dorsomedial part contains no immunoreactive cells and few SP-IR fibers. A high density of SP-IR nerve fibers and moderate density of SP-IR perikarya is seen ventromedially in the arcuate nucleus.

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Figs. 35-37. Fig. 35: Low power photomicrograph from t he caudal portion o f th e VMH. A high density of immunoreactive elements is seen in the ventrolateral part of t he nucleus (open curved arrows). Scale bar = 200 k m. Fig. 36: High power photomicrograph of a mediumsized SP-IR perikaryon in t h e ventrolateral part of t h e VMH. Scale bar = 50 pm. Fig. 37:Medium power photomicrograph showing the

midportion of the arcuate nucleus. A number of small SP-IR somata is seen within the borders of the nucleus (solid arrows). Note the low density of immunoreactive material in the neurohemal region of the adjacent median eminence in this section from a colchicine-treated animal (open curved arrow). Scale bar = 100 km.

magnocellular nucleus (TMC), the density of SP-IR fibers decreased significantly (Fig. 5C). The perifornical nucleus is dorsocaudally continuous with the submammillothalamic nucleus (SMT), which contained a low to moderate density of SP-IR fibers and terminals. From the SMT, few stained fibers could be followed ventrally into the perifornical area, while others entered the mammillothalamic tract. Further caudally, the supramammillary nucleus (SUM) was seen to contain a high density of SP-IR fibers (Fig. 5D). Laterally, immunoreactive fibers could be followed coursing into the medial forebrain bundle, while at a more caudal level the majority of SP-IR fibers coursed dorsally into the PH. Thus, the stained fibers from the SUM merged with

those of the P H to course into the periventricular fiber bundle of the third ventricle. Within all subnuclei of the medial mammillary nucleus, SP-IR fibers were sparse. SP-IK cell bodies. The rostra1 part of the mammillary region contained a high number of SP-IR cells. Throughout the rostrocaudal extent of the PH, a high number of diffusely scattered SP-IR cell bodies was observed (Fig. 45). The cell bodies were either small, ovoid, or medium-sized, and multiform. A moderate number of small and round cells was observed in the PMV (Fig. 44). From the perikarya of some of these cells immunoreactive axons could be followed over a short distance into the PMD. In the PMD, a very large

SUBSTANCE P IN THE HYPOTHALAMUS

Figs. 3841. Fig. 38: Low power photomicrograph from a noncolchicine-treated animal showing t h e midportion of the DMH. A low density of SP-IR fibers and terminals is seen in t h e central part of the nucleus (solid arrow). In contrast, a very high density of immunoreactive fibers is seen in the adjacent diffuse part of t h e nucleus and in particular in the ventrolateral border zone between the two subnuclei (open curved arrow). Scale bar = 200 pm. Fig. 39: Low power photomicrograph from a colchicine-treated animal showing t h e DMH at a level corresponding to t h a t shown in Figure 38. The central part of the DMH (solid arrow) is devoid of immunoreactive somata, while the

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surrounding diffuse part contains a high number of medium-sized SP-IR perikarya. The dense SP-IR fiber plexus shown in Figure 38 does not disappear after colchicine treatment (open curved arrow). Scale bar = 200 wm. Fig. 40: Medium power photomicrograph from t h e caudal part of the DMH. Except for t h e medially situated central part (open curved arrow), a high density of medium sized SP-IR perikarya is seen throughout the nucleus. Scale bar = 100 pm. Fig. 41: High power photomicrograph showing multiform, medium-sized SP-IR neurons of t h e DMH. Scale bar = 20 pm.

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Figs. 42-45. Fig. 42: Low power photomicrograph from t h e premammillary region. A high density of SP-IR elements is seen within the border of the ventral premammillary nucleus (solid arrows). From the PMV a dense plexus of SP-IR fibers can be followed to the dorsal premammillary nucleus (open arrows). Scale bar = 500 pm. Fig. 43: High power photomicrograph showing a large SP-IR perikaryon from the tuberal magnocellular nucleus. Scale bar = 20 pm. Fig. 44: Low power photomicrograph from the caudal part of the premammillary

P.J. LARSEN

nuclei. A high density of SP-IR nerve fibers and terminals is seen in the PMV (open curved arrows), while the PMD is dominated by a high density of SP-IR cells (solid arrows). Scale bar = 200 p.m. Fig. 45: Low power photomicrograph showing a high density of small-sized SP-IR perikarya in the suprammillary nucleus (open curved arrows) and medium sized SP-IR perikarya in the posterior hypothalamic area (solid curved arrows). Scale bar = 200 pm.

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SUBSTANCE P IN THE HYPOTHALAMUS number of medium-sized, multiform SP-IR perikarya was observed (Fig. 44). Laterally to the PMD, in the SMT a moderate number of small, faintly stained cell bodies was seen. Within the magnocellular nuclei of the mammillary region, a low number of SP-IR perikarya was observed in the TMC. These cells were large and multiform although with poorly defined dendritic arbors (Fig. 43). Further caudally within the SUM,a large number of densely packed small and round perikarya was seen (Fig. 45). Axons from the SUM left the nucleus either laterally into the mfb or dorsally into the PH.

DISCUSSION Immunoblotting, preabsorption, and the fact that both antisera gave rise to identical staining patterns strongly suggest that the immunohistochemistry is specific for substance P. However, a remote possibility exists that a heretofor unknown antigen was visualized. In colchicinetreated animals, a high number of immunoreactive perikarya was observed in the full rostrocaudal extent of the preoptic area and the hypothalamus. Common problems associated with colchicine treatment are differential distribution of colchicine after intraventricular infusion and possible influence on gene expression. Thus, the number of SP-IR perikarya observed in the individual brain areas may not reflect the actual number of SPergic neurons and therefore quantification was not carried out. The findings of the present study are in general agreement with those of previous immunohistochemical and in situ hybridization studies (Ljungdahl et al., '78; Cuello and Kanazawa, '79; Panula et al., '84; Warden and Young, '88; Harlan et al., '89). However, by taking into account more recent information about the morphological and functional organization of the preoptic area and hypothalamus of the rat, the present results extend considerably what is known about the organization of SPergic systems within these areas. The combination of colchicine treatment and a highly specific antiserum revealed a higher number of immunoreactive perikarya than earlier reported in the hypothalamus. Furthermore, areas previously claimed to be devoid of SP-IR cell bodies turned out to contain such neurons-in some instances even in substantial numbers. The use of the avidin-biotin system may have contributed to the revelation of further hypothalamic SPergic structures, since this method is usually more sensitive than immunofluorescent and peroxidase-antiperoxidase techniques. In the followingparagraphs, some comments on the possible connectivities of the hypothalamic areas containing SP-IR elements area are given.

Preoptic area The presence of SP-IR cell bodies in the medial preoptic nucleus (MPN) is debatable since somewhat discrepant results exist. Simerly et al. ('86) described a low number of SP-IR cell bodies in all subsets of the MPN although they predominated in the lateral part of the MPNm. In contrast to this, Panula et al. ('84) reported that SP-IR cells are concentrated dorsally in an area corresponding to the strial part of the MPO. The present findings clearly show that SP cell bodies are rare within the MPN proper, whereas a high number of stained perikarya are present in the surrounding MPO. The immunoreactive perikarya observed in the MPN of the former study may have been gastrin-releasing peptide (GRP) neurons since the antiserum employed was

reported to cross-react with bombesin. GRP shares considerably sequence homology with substance P (Moody et al., '81; Larsen et al., '89a), leaving the specificity of SP immunoreactivity in many studies questionable. The distribution of SP-IR fibers in the MPN is largely consistent with that described earlier (Simerly et al., '86), although the observed shift in fiber density along the rostrocaudal axis has not been reported previously. The MPN receives projections from widespread regions of the brain, and these projections are topographically distributed within the three subdivisions of the nucleus (Conrad and Pfaff, '76; Simerly and Swanson, '86). Most of the SP-IR fibers probably reach the MPN via the periventricular route. Accumulated knowledge from tracing experiments and immunohistochemicalstudies suggest that hypothalamic SP-fierents to the MPN could have their origin in the mpPVN, VMHVL, DMH, and supramammillary and ventral premammillary LH area nuclei, whereas limbic SPergic inputs could come from BSTe, BSTp, medial and posterior amygdala, and lateral septum (ventral part). The ventrolateral part of the VMH sends ascending projections via the periventricular route and the anterior hypothalamic area to enter the caudal pole of the MPN. The distribution of SP-IR fibers in the MPN subnuclei corresponds to that seen for anterogradely labeled VMH terminals (Simerly and Swanson, '86). Furthermore, Yamano et al. ('86) demonstrated that SP-IR cell bodies in the VMH were retrogradely labeled after large injections of tracer into the MPO, suggestingthat VMHVL contributes with an SP input to the medial and lateral part of the MPN. Paxinos et al. ('78) suggested that the MPO receives an SPergic input from the BST based on a combined lesion and immunohistochemicalstudy. The lesions of that study may have involved additional afferents from the ventral part of the lateral septum-a region containing a high density of SP-IR perikarya (Ljungdahl et al., '78). The course of the periventricular SP-IR fibers suggests that fibers in the AVPv may be afferentsfrom the lateral septal area (Wiegand, '84; Simerly and Swanson, '87). The plexus of SP-IR fibers seen in the parastrial nucleus could originate from the DMH, which is known to innervate this nucleus densely (Simerlyand Swanson, '86).

Anterior region of the hypothalamus The distribution of SP-IR fibers and terminals in the suprachiasmatic nucleus (SCN) closely corresponds to the part receiving afferents from retinal ganglion cells (Moore and Lenn, '72) and the ventral lateral geniculate nucleus (Swanson et al., '74; Card and Moore, '82). SP-IR nerve fibers have been demonstrated within the optic nerve (J.D. Mikkelsen, personal communication) and it is possible that SP is a neurotransmitter involved in the retinohypothalamic tract. The moderate number of SP-IR somata observed in the SCN may themselves contribute to the intranuclear stained fibers. The number of immunoreactive cells seen in the present study is considerably higher than those earlier reported (Ljungdahl et al., '78; Panula et al., '84). The efferent projections of the SCN are mainly directed to the contralateral SCN, SubPV, PVN, MPO, lateral septal nuclei, paraventricular thalamic nucleus, and lateral geniculate nuclei (Watts et al., '87). However, the topographical distribution of SP-IR neurons leaving the SCN is not as obvious as, for instance, that of GRP (Mikkelsen et al., '91).The dorsal orientation of a moderate number of SP-IR nerve fibers apparently leaving the SCN

308 suggests that at least part of the high number of SP-IR terminals observed in SubPV may originate in the SCN. The midline area intervening between the two SCN nuclei was heavily innervated, which may reflect that the SPergic SCN neurons are in fact interneurons bilaterally synchronizing the activity of other SCN neurons. Electron microscopic studies show that the majority of synaptic interactions in the PVN are axodendritic rather than axosomatic (Silverman et al., '82). Furthermore, it is evident from Golgi studies and immunocytochemical staining of the PVN that dendritic arbors of magnocellular and parvicellular neurons are not restricted to the boundaries of individual subnuclei (van den Pol, '82). Thus, the highest density of axon terminals belonging to limbic afferents from the lateral septal area, the BST, and the central and medial amygdaloid nuclei to the PVN are found in the areas immediately surrounding the nucleus (Oldfield et al., '85; Gray et al., '89). In contrast, ascending noradrenergic inputs from the A1 are strictly confined to the outline to the pmPVN (Sawchenko et al., '85; Cunningham and Sawchenko, '88). Recently, an extensive retrograde tracing study has confirmed that the majority of SP-IR afferents in the PVN complex arise from perikarya in the lateral hypothalamic area, the BST, the laterodorsal tegmental nucleus, and the brain stem catecholaminergic C1 and A1 areas (Bittencourt et al., '91). Although only a small proportion of brain stem catecholaminergic neurons co-store SP, these neurons are likely to represent the major input to magnocellular somata since brain stem transection at the medullopontine level severely depletes the number of SP-IR terminals in the magnocellular PVN and SON (Bittencourt et al., '91). The most prominent limbic input to the magnocellular PVN originates in the BST and, furthermore, there is a high degree of correspondence between the autoradiographic pattern of terminals seen after anterograde L3H1amino acid tracing and that of SP-IR terminals demonstrated in the present study (Sawchenko and Swanson, '83). In particular, this is the case for the pmPVN, where a low density of terminals was present within the subnucleus itself, whereas a high density of terminals was present in the dorsolateral perimeter of the PVN. From here, the stained fibers could be followed laterally towards the fornix; scattered along this trajectory are axons and dendrites of magnocellular neurons (van den Pol, '82). Thus, this area may represent a location where SPergic axons could influence vasopressinergic magnocellular neurons. This result is in contrast to what has been found in the mouse (Stoeckel et al., '82) and it is not altogether supported by an electron microscopic double-labeling study demonstrating axosomatic synapses between SPergic terminals and vasopressinergic cell bodies (Heike et al., '86). However, further studies are needed to clarify the importance of possible axodendritic and/or axo-axonic synapses between SPergic terminals and magnocellular neurons in the immediate surroundings of magnocellular PVN subnuclei. The parvicellular subnuclei of the PVN receive afferents from most other hypothalamic cell groups (Sawchenko and Swanson, '83; Ter Horst et al., '87). Strikingly, most of these intrahypothalamic projections each end preferentially, and differentially, in more than one of the five distinct subnuclei. Possible SPergic afferents could arise in MPO, LPO, AVPv, SCN, caudal arcuate, VMHVL, DMH, ventral premammillary and supramammillary nuclei, and the retrochiasmatic, lateral, and posterior hypothalamic areas. Employing true blue as a- retrograde tracer, SP

PJ. LARSEN perikarya localized in a large proportion of these areas have recently been shown to project to the PVN complex (Bittencourt et al., '91). Of particular interest is the DMH projections to most of the parvicellular subnuclei of the PVN (Ter Horst et al., '87). The DMH most heavily innervates the dpPVN, IpPVN, and ventral part of the mpPVN; these subnuclei were indeed the ones that demonstrated the highest density of SP-IR terminals. The vast majority of the dpPVN neurons project to the intermediolateral column of the spinal cord, while most cells in the ventral part of the mpPVN and lpPVN preferentially project to the dorsal vagal complex and/or the spinal cord (Hosoya, '80; Swanson and Kuypers, '80; Swanson et al., '86). SP terminals in these PVN subnuclei are in a position to influence autonomic functions, either directly via SPergic neurons projecting to the lower brain stem and the spinal cord, or via SPergic inputs to the PVN, for instance from the DMH. The number of interneurons in the PVN complex is very high (van del Pol, '82). Thus, many of the SP-IR terminals observed within the borders of the PVN complex could have their origin from parvicellular PVN perikarya. The presence of SP in magnocellular hypothalamopituitary neurons has earlier been reported in the mouse but never in the rat (Stoeckel et al., '82). In that study it was suggested that SP is colocalized with vasopressin-a suggestion in agreement with the topographical distribution of magnocellular SP neurons in the present study. Until now, no in situ hybridization study has described the presence of preprotachykinin-A mRNAs in the magnocellular subnuclei of the PVN and SON. The reason for this discrepancy is at present unknown and it would be of future interest to clarify which physiological stimuli-if anycould trigger transcription of the preprotachykinin-A gene in magnocellular neurons. Otherwise, the presence of SP in magnocellular neurons could reflect receptor-mediated endocytosis of the ligand as described for SP bound to anterior pituitary cells (Larsen et al., '89c). However, the dense SP innervation observed in the posterior pituitary lobe strongly indicates that SP is a transmitter substance synthesized within magnocellular hypothalamo-pituitary neurons.

Lateral hypothalamic area Tracing experiments elucidating the connectivity of the lateral hypothalamic area have always been difficult to interpret because fibers of the medial forebrain bundle pass through it. As mentioned above, SP-IR neurons localized in the ventral portion of the lateral hypothalamic area have been shown to contribute with a substantial input to the PVN complex (Bittencourt et al., '91). Some evidence has accumulated that SP-IR neurons of the lateral hypothalamic area project to the parabrachial area of the pons (Milner and Pickel, '86). In addition, neurons of the lateral hypothalamic area send projections to the spinal cord (Hosoya, '80). Most of these perikarya are located in the dorsal LH and the perifornical region which in the present study contained a moderate number of SP-IR somata. Thus part of the SP-IR neurons in the perifornical nucleus may send projections to the spinal cord, which is heavily innervated with SP-IR terminals (Ljungdahl et al., '78).

Region of the tuber cinereum The SP-IR cell group of the ventrolateral part of the ventromedial hypothalamic nucleus (VHMVL) is probably one of the most widely described SP-containing entities of the hypothalamus (Akesson and Micevych, '88). In accord

SUBSTANCE P IN THE HYPOTHALAMUS

309

The immunoreactive fibers within the external zone of the ME were preferentially observed in the palisade layer. As seen from the free-floatingsections, the SP immunoreactive material observed in the ME of nontreated animals was not strictly confined to neuronal elements. The diffuse SP-IR staining appeared to be located within the communicating perivascular and subendothelial extracellular space surrounding the neuroendocrine terminals and tanycyte processes. A similar labeling of the neurohemal region of the ME has earlier been reported by Krisch and Leonhardt ('78). Based on ultrastructural HRP-labeling studies these authors concluded that a border area exists adjacent to the dorsolateral aspect of the neurohemal region of the ME, where tanycytes isolate the ME neuropil from the neighboring areas. This finding may indicate that SP released in the external zone of the ME influences the activity of neighboring neuroendocrine terminals. A direct action of SP on ME elements is further supported by the presence of a high density of autoradiographically demonstrable 1251-Bolton Hunter SP binding sites in the ME (unpublished observation). The possible origins of SP-IR terminals in the external zone of the ME are at present only speculative. Retrograde tracing experiments from the ME have shown labeled somata in the MPO, SON, parvicellular PVN, PE, Arc, and VMH (Wiegand and Price, 'SO), and all of these regions could contribute SP-IR afferents to the ME. From a lesion experiment it has been suggested that the Arc is the major source of SP-IR terminals in the ME (Palkovits et al., '89). However, the relative absence of SP-IR perikarya in the Arc and the low number of SP-IR fibers penetrating into the ME from here makes it likely that also neurons from other hypothalamic nuclei may contribute SP-IR fibers and terminals in the ME. A possible origin for such a projection is the mpPVN (Larsen et al., '91).This is further supported by a study in which rats were given monosodium glutamate neonatally, a treatment known to lesion the arcuate neurons (Jessop et al., '91a). In that study, the content of ME-immunoreactive SP was unaffected by the treatment. Otherwise, the SP-IR somata of the Arc may be regarded as interneurons influencing the activity of yet other Arc neurons containing ACTH (Hisano et al., '84), metenkephalin (Tsuruo et al., '831, growth hormone releasing factor (Daikoku et al., '881, or even SP (Tsuruo et al., '84). Some of the SP-IR nerve fibers in the internal zone of the rostral part of the ME could be followed into the infundibular stalk, suggesting that the SP-IR fibers and terminals observed in the PP course via the rostral ME to this distal location. As was the case for the ME, a much higher number of SP-IR elements was observed in the PP in the present study than previously claimed. In contrast, a high density of SP-IR nerve fibers and terminals within the PP has been described in the mouse (Stoeckelet al., '82). The presence of two types of SP-IR terminals in the PP could indicate that SP exerts a dual functional role within the organ. Thus, SP could be released from large peptidergic terminals to the Median eminence, posterior pituitary lobe, and systemic blood circulation as well from smaller terminals exerting a local function within the PP. However, a receptor arcuate nucleus binding study performed on isolated PP membranes has The presence of SP-IR nerve fibers in the median eminence (ME) and the posterior pituitary lobe (PP) has not been able to demonstrate specific SP binding sites above the assay detection limit (Larsen et al., '89d). previously been described in several mammalian species (Hokfelt et al., '78; Rprnnekleiv et al., '84; Stoeckel et al., Mammillary region '82; Tsuruo et al., '83; Mikkelsen et al., '89). However, the immunohistochemical procedure presently used revealed a Within the mammillary region, the mammillary nuclei much higher number of SP-IR nerve fibers in the ME and proper were nearly devoid of SP-IR material, while the PP than earlier reported in the rat. mammillary fiber capsule and the surrounding supramam-

with earlier immunohistochemical and in situ hybridization studies, the present study shows that within the VMH the SPergic somata are largely confined to the ventrolateral part (Milner and Pickel, '86; Warden and Young, '88; Harlan et al., '89). As seen in the present study and in details from Golgi impregnation studies (Millhouse, '731, the perikarya of the VMHVL send out their dendritic arbors to the ventrolateral perinuclear shell. From other hypothalamic nuclei, this area receives af€erents from the anterior hypothalamic area, ventral premammillary nucleus, and dorsomedial nucleus (Conrad and Pfaf€, '76; Ter Horst and Luiten, '87). As seen from the present study, the former of these projections is unlikely to be SPergic, while the two latter may contribute to the high density of SPergic terminals seen among the SP-IR dendrites in the perinuclear shell of the VMH. In terms of functional relations to the SPergic VMHVL neurons, it is of interest to know whether these are influenced by endogenous or incoming SPergic terminals. Otherwise the SPergic perikarya could be influenced by the enkephalinergic cells of the VMHVL (Dupont et al., '80) similar observations made in the dorsal column of the spinal cord (Mudge et al., '79). Anatomical tract tracing studies have shown that the VMH neurons send out intrahypothalamic projections to rather few hypothalamic nuclei, among which are the DMH, dorsal premammillary, MPO, MPN, and hypothalamic periventricular nuclei (Kita and Oomura, '82; Yamano et al., '86; Ter Horst et al., '87). In the present study SP-IR axons could be followed from the VMHVL coursing dorsomedially to the DMH. However, the DMH is reciprocally innervated with the VMH, leaving the possibility open that these fibers may originate from SPergic cell bodies within the diffuse part of the DMH. Finally, the relatively high density of SP-IR fibers and terminals seen in the dorsal premammillary nucleus (PMD) may be of VMH origin, although a great number of the stained fibers in the PMD appeared to enter this nucleus from the ventrally situated ventral premammillary nucleus. The VMHVL sends descendingprojectionsvia the periventricular strata to the periaqueductal grey (PAG) (Conrad and Pfaff, '76; Luiten et al., '85). As discussed later, it has been suggested that at least some of the descending VMH neurons are SPergic neurons involved in the facilitation of lordosis and sexual receptivity in the female rat (Dornan et al., '87, '90). However, large unilateral lesions of the VMH do not reduce the intensity of SP-IR staining in the PAG, which may indicate that VMH afferents are not the major source of SPergic terminals within the PAG (Malsbury, '85). Finally, the VMH establishes numerous efferent connections within the borders of the nucleus itself, and many of these synaptically interconnect individual VMH units (Millhouse, '73; Nishizuka and Pfaff, '89).

PJ. LARSEN millary and premammillary nuclei were heavily innervated with SP-IR terminals. These nuclei contained a high number of stained perikarya. Anatomical tract tracing studies have shown that the relatively SP-IR fiber-rich mammillary fiber capsule receives input from basal forebrain structures, such as the BST, the MPO, the area of the tuber cinereum, and the border zone between the lateral and medial hypothalamic areas (Conrad and PfafT, '76; Shibata, '89). All of these hypothalamic areas could convey SPergic d e r e n t s to the mammillary fiber capsule and the supramammillary and dorsal premammillary nuclei, although other sources obviously exist. The ventral premammillary nucleus and the VMH are heavily innervated from the medial and corticomedial amygdaloid nuclei (Luiten et al., ' 8 9 , and the medial amygdala is known to contain a high number of SP-IR perikarya (Cassell and Gray, '89). The majority of these fibers course via the stria terminalis, which contains a high number of SP-IR fibers. Accordingly, the medial amygdala may in part contribute with a SPergic input to the ventral premammillary nucleus. The target areas of the efferent projections from the nuclei of the mammillary region containing SP-IR perikarya are at present mostly speculative. However, evidence has accumulated that the supramammillary region and the posterior hypothalamic area send SP-IR axons to the hippocampal formation and entorhinal cortex (Gall and Selawski, '84; Ino et al., '88; Yanagihara and Niimi, '89). The functional importance of such a supramammillaryhippocampal projection is unknown, but based on the discrete distribution of SP-IR fibers within the hippocampal subregions it has been suggested that the tachykinins exert a selective localized function (Shults et al., '87). The terminal field of the small population of magnocellular SP-IR neurons in the tuberal mammillary nucleus is at present not thoroughly known. Combined tracing and immunohistochemical experiments have shown a small number of retrogradely labeled SP-IR cells in the TMC after injection of tracer into the septa1 region and amygdala (Kohler et al., '85). However, the connectivity of the TMC is far more complex, and many possible target areas exist for the magnocellular SP-IR neurons.

and recently an SP-containingprojection from the VMHVL to the dorsal PAG has been demonstrated (Dornan et al., '90). However, preprotachykinin-A gene expression in these cells appears to be unaffected by estrogen treatment (Romano et d., '87, '88). When injecting SP into the PAG, lordosis is facilitated in the female rat (Dornan et al., '87), and the concentration of SP in this area varies with the estrous cycle (Frankfurt et al., '86). The influence of SP on anterior pituitary hormones involved in the reproductive cycle is somewhat complicated. Not much is known about the SPergic innervation of hypothalamic cells producing and releasing hypothalamic factors, but the hypothalamic content of SP, preprotachykinin mRNA, staining intensity, and number of SP-IR perikarya in the arcuate nucleus are related to the estrous cycle, suggesting an influence on gonadal hormones (Kerdelhue et al., '85; Tsuruo et al., '87; Brown et al., '90). Injection of SP into the third ventricle is reported to stimulate the release of LHRH into the portal circulation (Vijayan and McCann, '791, while SP has an inhibitory action on the LH release from anterior pituitary cells at some states in the estrous cycle (Kerdelhue et al., '85).Morphological evidence for such a n action is the recent demonstration of SP-IR terminals-presumably from arcuate neurons-synaptically contacting LHRH-containing perikarya in the preoptic area (Tsuruo et al., '91). Substantial evidence has accumulated that SP influences the anterior pituitary secretion of prolactin (Kato et al., '76; Rivier et al., '77; Chihara et al., '78; Vijayan and McCann, '79; Abe et al., '81; Eckstein et al., '80). SP acting in the anterior pituitary could either be secreted from pituitary cells themselves or originate from external sources such as the ME (Aronin et al., '86). The concept of a direct action is further supported by the presence of a membrane-localized Nk- 1 receptor coupled to phospholipase C-mediated polyphosphoinositide metabolism on anterior pituitary cells (Larsen et al., '89b,c; Mau et al., '90). The presence of perivascular SP-IR fibers in the external layer of the ME and the posterior pituitary lobe may indicate that SP is conveyed from the hypothalamus to the anterior pituitary by a vascular route (Tsuruo et al., '83, '87; Mikkelsen et al., '89). However, the assumption that the relatively large amounts of SP located in the external zone of the ME are Functional implications released to act at the level of the anterior pituitary can not The presence of a high density of SP-IR perikarya and be sustained in the absence of evidence for higher concentranerve fibers in many hypothalamic nuclei and areas sug- tions of SP in hypophysial portal blood than in the periphgests that SP is involved in a variety of hypothalamic eral circulation (Eckstein et al., '80; Lim et al., '90). Water homeostasis. A role for SP as a neurotransmitter functions. Although it is beyond the scope of the present paper to comment on all possible actions, some observa- in the regulation of the magnocellular hypothalamopituitary system has been suggested (Sladek and Armtions are given on selected functional systems. Reproductive behavior. Several lines of evidence have strong, '87). It is well known that sodium loading or indicated that SP influences the reproductive state and dehydration lead to increased secretion of neurohormones sexual behavior in male as well as female individuals. In the from the posterior pituitary lobe (Jones and Pickering, '69). male rat, injections of SP in the MPO significantly reduce Within the posterior pituitary lobe of sodium-loaded rats, the latency to initiate copulatory activity while SP injec- the concentration of SP is lowered about 70% (Holzbauer et tions in the lateral ventricle have the opposite effect al., '84). The immunohistochemical findings of the present (Dornan and Malsbury, '87, '89). As mentioned, it has been study strongly suggest that SP is involved in posterior suggested that the ventrolateral part of the VMH is the pituitary function either by influencing other peptidergic main source of SP-IR fierents in the MPO (Yamano et al., terminals and/or pituicytes like the opioids (Lightman et '86). Interestingly, the VMHVL is partly involved in the al., '83), or by being directly released to the systemic hormonal induction of female rodent receptivity via projec- circulation. In favor of the latter suggestion is the apparent tions to the PAG (Sakuma and Pfaff, '79; Manogue et al., absence of SP binding sites on posterior pituitary mem'80; Morel1 and PfafT, '82; Dornan et al., '87). The idea that branes (Larsen et al., '89d). Intraventricular injections of SP have a profound and SP is actively taking part in this action is supported by the finding that parts of the estrogen-concentratingcell bodies long-lasting effect on the release of arginine-vasopressin in the V H W are SPergic (Akesson and Micevych, '88), (aVP) to the systemic blood circulation (in addition to

SUBSTANCE P IN THE HYPOTHALAMUS lowering the urine flow) (Chowdrey et al., '90). The site of action of SP on a VP release is at present unknown, but in addition to a direct influence on magnocellular aVPcontaining neurons, the subfornical organ may be involved in this response. The subfornical organ contains a high density of Nk-1 receptors (Saffroy et al., '88) and the neurons from this area directly project to the magnocellular PVN and supraoptic nuclei (Miselis et al., '79). Apart from a direct stimulatory action on the aVP release and thus the renal water conservation, SP has profound effects on the behavioral aspect of water homeostasis by being a potent antidipsogenic substance in rodents (de Car0 et al., '80; Fitzsimons and Evered, '80; Massi et al., '88).In the rat, SP and other tachykinins significantly reduce angiotensin-11induced drinking, suggesting that the subfornical organ, which has a high content of angiotensin-I1 binding sites, may be involved in this rather peculiar action.

ACKNOWLEDGMENTS I thank Drs. J.D. Mikkelsen and M. Maller for advice and support, Dr. J.J. Holst for supplying the antiserum, Ms. B. Houlind and Ms. G. Hahn for excellent photographic assistance, and Mrs. L. Olsen for excellent technical assistance. Support for the study was given by NOVO's Fond, Carl P. Petersens Fond, Fonden ti1 Legevidenskabens Fremme, and Axel Thomsen og hustru Martha Thomsen's Fond.

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Distribution of substance P-immunoreactive elements in the preoptic area and the hypothalamus of the rat.

The localization and morphology of neurons, processes, and neuronal groups in the rat preoptic area and hypothalamus containing substance P-like immun...
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