Immuno-enzyme cytochemical demonstration of mesotocinergic nerve fibres in the pars intermedia of the amphibian hypophysis

Summary Immuno-enzyme cytochemical investigations showed that the whole amphibian pars intermedia of the hypophysis is innervated by an intercellular network of peptidergic varicose nerve fibres which contain mesotocin or (and) parts of the mesotocin molecule. The pars intermedia does not contain vasotocinergic fibres. The mesotocinergic fibres are branches of axons leaving the pituitary stalk and the neural lobe. In animals of which the hypothalamic magnocellular neurosecretory preoptic nuclei had been completely removed, the immuno-reactive mesotocinergic fibres of the pars intermedia had totally disappeared. From this result, it is concluded that the mesotocinergic fibres of the pars intermedia of the amphibian hypophysis are axons of neurosecretory perikarya located in the hypothalamic magnocellular neurosecretory preoptic nuclei.Dedicated to Professor Berta Scharrer on the accasion of her 70th birthdayThis investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

The pars intermedia of the mink,Mustela vison

Summary The pars intermedia of intact and experimental female minks has been studied by light, electron and fluorescence microscopy. The general structure of the mink intermediate lobe is described. Two main cell types are recognized. One, termed glandular cell, predominates in number and is characterized by the presence of electron-dense granules about 200 nm in diameter and numerous large vesicles up to 300 nm in diameter. The other, termed stellate cell, is devoid of cytoplasmic vesicles and granules and possesses microfilaments, junctional complexes and elongated processes inserted between the glandular cells. Different treatments (photostimulation and administration of hypertonic saline and metopirone) induced morphological reactions in the glandular cells. The significance of these changes and the possibility of a functional relation between the pars intermedia and ACTH secretion are discussed.Numerous nerve fibres and axon terminals containing electron-dense granules (60–120 nm) and electron-lucent vesicles (30–40 nm) are observed throughout the pars intermedia.With the histochemical fluorescence method of Falck-Hillarp a rich system of delicate fluorescent varicose fibres, sometimes provided with irregular swellings or droplets, is observed in the pars intermedia and also in the pars nervosa. Microspectrofluorometrically these fibres exhibit the spectral characteristics of catecholamines. All the cells of the pars intermedia and a large number of cells in the pars distalis show a yellowish weak fluorescence, which becomes much stronger after combined formaldehyde-ozone treatment. This cellular fluorophore shows the same microspectrofluorometric characteristics as does the fluorophores of tryptamine and of certain peptides with NH₂-terminal tryptophan.Supported by the Swedish Fur Breeders' Association and the Swedish Natural Science Research Council (grant No. 2124). Thanks are due to Miss W. Carlsson and Miss Y. Lilliemarck for their helpful technical assistance.Supported by the Harald and Greta Jeanssons Stiftelse and by the Ford Foundation. The skilful technical assistance of Mrs. Eva Svensson and Miss Annika Borgelin is greatfully acknowledged.     

Pituitary monoamines of the cat with special reference to the presence of an unidentified monoamine-like substance in the adenohypophysis

Summary In the pituitary gland of the cat, dopamine (M.V. 0.78 g/g), noradrenaline (M.V. 0.29 g/g) and 5-HT (M.V. 0.94 g/g) have been found. With the histochemical fluorescence method, a rich system of delicate fluorescent varicose fibres, often provided with irregular swellings or droplets, was observed in the neural lobe and pars intermedia. Microspectrofluorimetrically, these fibre structures exhibit the spectral characteristics of catecholamines. Most cells in the pars intermedia and a large number of cells in pars distalis show a yellowish fluorescence, with microspectrofluorimetric characteristics which differ entirely from those of the catecholamines and 5-HT. In animals treated with reserpine, the pituitary dopamine, noradrenaline, and 5-HT are largely depleted. However, the intensity and the spectral properties of the cellular fluorescence are not affected by this treatment, whereas the fluorescent fibres can no longer be seen. Thus none — or only little — of the catecholamines and 5-HT but some other monoamine-like substance is stored in the fluorescent cells of the adenohypophysis. Preliminary studies suggest that this substance is closely related to or perhaps identical with tryptamine.Supported by grants from the Swedish Medical Research Council (No B 68-12X-712-03 B and B 68-14X-56-04 B), the Association for the Aid of Crippled Children, New York, and by the Faculty of Medicine, University of Lund, Sweden.     

Existence of noradrenalin cells and serotonin cells in the pituitary gland of Rana catesbeiana

The localization of catecholamine-synthesizing enzymes [tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine-N-methyltransferase (PNMT)], of serotonin (5-HT), and adrenocorticotropin (ACTH) in the pituitary of bullfrog (Rana catesbeiana), rat, hamster, and dog was examined by the immunofluorescence method. Many TH- and DBH-positive but PNMT-negative noradrenalin cells and 5-HT-positive serotonin cells were first observed in the pars distalis of the frog pituitary together with small numbers of ACTH-positive cells, in marked contrast to our previous findings that TH-positive but DBH-negative dopamine cells are rare in the anterior lobe of rat and dog pituitary. The entire population of cells of the pars intermedia showed a weak ACTH-like immunoreaction. Although most of these cells were TH- or 5-HT-negative, TH-positive but DBH-negative dopaminergic varicose fibers surrounded these cells. Among cells of the pars intermedia of the frog, 5-HT-positive cells with processes were also scattered. In the neural lobe, TH- and 5-HT-immunoreactive fibers were rarely seen.     

Monoamines in the hypothalamo-hypophyseal system of the mouse with special reference to the ontogenetic aspects

Summary An accumulation of monoamines is found in the adult eminentia mediana and infundibular stem, close to the capillaries of the primary plexus, where they seem to be stored mainly in closely packed delicate varicose fibres, often with swollen endings, droplets. The first traces of monoamines are visible in the embryonic eminentia of fetuses from the last three days of the gestation period, but typical monoamine-containing fibre endings close to the primary plexus capillaries in zona externa are not found until towards the end of the first post-natal week. The adult condition is attained two weeks later. Nearly all cells in the adult pars intermedia show a specific yellowish fluorescence whose intensity shows a wide individual variation. Such fluorescent cells are sometimes present in the intermedia of the new-born. In some but not all adult specimens, a fluorescent varicose fibre system is visible. Fluorescent fibres were first observed in the intermedia of the new-born. Fluorescent fibres are visible in n. arcuatus and n. paraventricularis in the fetus at the 16th day of the gestation period. Nerve cell bodies storing visible amounts of monoamines are found in n. arcuatus, n. periventricularis anterior, and n. premammillaris ventralis of the adult. This amine storing activity is first visible in n. arcuatus of the embryo at the 17th day of the gestation period. The adult condition of these cells is reached during the first postnatal week.     

The localization of oxytocin,vasopressin, somatostatin and luteinizing hormone releasing hormone in the rat neurohypophysis

Summary The hypothalamic hormones arginine-vasopressin (AVP), oxytocin (OXT), somatostatin (SOM), and luteinizing hormone-releasing hormone (LHRH) were localized in the rat neurohypophysis by the use of semithin serial sections and the unlabeled antibody enzyme method. Clusters of AVP fibres are present within the central region of the neural lobe, clusters of OXT fibres mainly in the peripheral part. The AVP fibres enter bilaterally into the neural lobe.The results call into question previous reports on the presence of AVP on receptors in the pars intermedia cells, since incubation with anti-AVP resulted in similar staining in the pars intermedia of the Wistar and homozygous Brattleboro rat, a mutant strain deficient in AVP. The same intermediate lobe cells are stained after incubation of serial sections with anti-AVP and anti--melanocyte-stimulating hormone (-MSH). This staining of anti-AVP could be removed by solid phase absorption to -MSH and is thus most probably due to cross reaction with -MSH. SOM fibres appear to be present in the peripheral parts of the proximal neurohypophysial stalk and mainly lateral in its more distal parts. In the neural lobe they rapidly decrease in number, although some fibres continue into the distal part of the neural lobe, running bilaterally and situated adjacent to the pars intermedia. The SOM staining within magnocellular elements, which has been reported in the literature, can most probably be explained by cross reaction of anti-SOM with neurophysins. LHRH fibres are very scarce in the neurohypophysial stalk and absent in the neural lobe.Supported by the Foundation for Medical Research FUNGOThe authors wish to thank Drs. J. De Mey (Beerse, Belgium), A. Arimura (New Orleans, U.S.A.), M.P. Dubois (Nouzilly, France), B.L. Baker (Ann Arbor, U.S.A.) and A.G.E. Pearse (London, U.K.) for their gifts of anti-somatostatin serum, Dr. B. Kerdelhué (Gif-sur-Yvette, France) for anti-LHRH serum, and Dr. F. Vandesande (Ghent, Belgium) for anti-neurophysin I and II serum and bovine neurophysin I and II. Dr. J.G. Streefkerk (Free University, Amsterdam) is acknowledged for critical comments and Mr. A.T. Potjer and Miss J. van der Velden for their skilled assistance     

Organization and ultrastructural identification of the catecholamine nerve terminals in the neural lobe and pars intermedia of the rat pituitary

Summary The central catecholamine innervation of the pituitary neural lobe and pars intermedia of the rat have been identified ultrastructurally and their organization has been investigated in a combined fluorescence histochemical and electron microscopical study. The dopamine analogues, 5-hydroxydopamine and 6-hydroxydopamine, were used to label the catecholamine terminals, and to enable the direct correlation between the fluorescence microscopical and the electron microscopical pictures.The fibre type that was identified as catecholamine-containing was ultrastructurally chiefly characterized by dense-cored vesicles, 500–1200 Å in diameter, intermingled with varying numbers of small empty vesicles. 5-hydroxydopamine was selectively accumulated in these fibres and caused an increased electron density of the granular vesicles as well as of some small normally agranular vesicles, and systemically administered 6-hydroxydopamine caused a selective degeneration of these fibres, most prominently within the neural lobe. The dopaminergic terminals of the neural lobe showed frequent close contacts (80–120 Å), without real membrane thickenings, to neurosecretory axons and to pituicyte processes. It is suggested that these close contacts might signify a direct dopaminergic influence on the neurosecretory axons and/or on the pituicyte processes. The identified central catecholamine fibres were also found to make common synapse-like contacts on the pars intermedia cells, whereas the innervation by neurosecretory fibres was very rare. This suggests that the direct central nervous control of the rat pars intermedia is exerted by the catecholamine neurons. A very special feature of the catecholamine fibres in the pituitary is the occurrence of peculiar, large dopamine-filled droplet-like swellings. Electron microscopically, such large axonal swellings (more than 2 in diameter) were found to contain, in addition to the characteristic vesicles and organelles, strongly osmiophilic lamellated membrane complexes resembling myelin bodies and multivesicular bodies encircling disintegrated vesicles, suggesting that these droplet fibres represent dilated stumps of spontaneously degenerating dopaminergic axons. It is suggested that the dopaminergic neural lobe fibres are undergoing continuous reorganization through degeneration—regeneration cycles, a phenomenon previously suggested for the neurosecretory axons of the neural lobe.Supported by the Deutsche Forschungsgemeinschaft.Supported by Svenska Livförsäkringsbolags Nämnd för Medicinsk Forskning, by The Medical Faculty, University of Lund and by the Ford Foundation.     

The arrangement of neurosecretory and catecholamine fibres in relation to the pituitary intermedia cells of the skate,Raja radiata

Summary The innervation pattern of the intermediate lobe of the skate (Raja radiata) was studied with histological and fluorescence histochemical methods. Neurosecretory fibres, stained with i.a. pseudo-iso-cyanine, were found running in bundles in the central parts of the cell cords. They terminated partly around the perinuclear parts of the intermedia cells, partly around the apices of the cells close to the vascular walls.A catecholamine innervation of the intermedia was also established. Catecholaminecontaining fibres with the appearance of nerve terminals were found around the intermedia cell apices close to the vessels. In some specimens, catecholamine fibres also seemed to terminate at the perinuclear parts of the cells.Thus it is possible, judging solely from structural relations, that both the cell body (the synthesis pole) and the cell apex (the release pole) receive a dual innervation. Recent experimental evidence indicates that the release of MSH from the pars intermedia is controlled by catecholamine fibres, but as yet there is only structural evidence for a special control of hormone synthesis.This study was supported by grants from the Swedish Natural Science Research Council (No. 99-35 and 2126-2) and was carried out within a research organization sponsored by the Swedish Medical Research Council (Projects No. B70-14X-712-05 and B70-14X-56-06).     

Localization of acetylcholinesterase and choline acetyltransferase in the rat pituitary gland

Summary Experiments were conducted to determine the presence of two cholinergic biomarkers, acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) in the rat pituitary. A histochemical procedure for AChE was used to provide visualization of structures containing this enzyme. Radiochemical methods provided a sensitive assay for measuring ChAT activity. Nerve fibres staining for AChE activity were observed in the neurointermediate lobe, with the greatest concentrations appearing at the junction region with the pituitary stalk. Cells staining for AChE were found in the pars distalis and pars intermedia. ChAT activity correlated well with AChE distribution in pars nervosa and pars intermedia but not in pars distalis. The greatest levels of ChAT activity were in pars intermedia and the region where the stalk joins the pituitary. Significant values were also found for the pars nervosa. The presence of AChE and ChAT in pars intermedia and pars nervosa is evidence for a cholinergic innervation to these regions. In pars distalis, where other investigators have found muscarinic receptors, intense staining for AChE and absence of ChAT activity may indicate non-innervated, acetylcholine-sensitive sites.     

Serotonin immunoreactivity in the intermediate lobe of the rat pituitary.

Immunocytochemical staining for serotonin (5-HT) in paraffin-embedded sections of rat pituitary resulted in the localization of reactive nerve fibres and cell bodies in the intermediate lobe. Immunostaining was also found in the anterior and posterior lobes. Labelled nerve fibres appear to enter the intermediate lobe from the neural lobe through the interlobular spaces. These fibres are relatively scarce and lightly stained. Neuroglandular contacts were identified between varicose nerve endings containing serotonin and immunoreactive perykarion. It is not clear whether intermediate lobe cells produced 5-HT themselves or, alternatively, these cells take in 5-HT from serotoninergic nerve terminals.     

Control of the pars intermedia of the lizard,Anolis carolinensis

Summary The capacity of colour change in intact lizards and in animals with a transected hypophysial stalk was studied during extended periods. It was concluded that, with certain exceptions, the skin colour of the lizards gives information on the circulating levels of MSH, and thereby on the function of the pars intermedia.After transection of the hypophysial stalk, three phases of chromatic behaviour were recognised. During Phase I, which lasted for about 6 days (average), dark skin was retained irrespective of the colour of the background (= high MSH levels), whereas Phase II (19 days) was characterised by the inability of the animals to become brown (= low MSH levels). A gradual recovery toward normal adaptive capacity was seen during Phase III.In the disconnected neuro-intermediate lobe, aldehyde fuchsin (AF)-positive material in the rostral region rapidly clumped and disappeared within a few days. In the caudal portion, the stainability and the varicose arrangement of fibres were retained longer, but disappeared within approximately 14 days after the operation. Proximal to the lesion, the preoptic system exhibited a marked increase in stainability with AF, starting in the median eminence and progressing in a retrograde direction toward the peptidergic nuclei.Revascularisation of the disconnected neuro-intermediate lobe occurred during the first few days. A reinnervation of AF-fibres across the transected area into the neural lobe was observed during Phase III in most animals, but AF-fibres did not reoccupy the entire lobe. In association with an outgrowth of capillaries, the regenerating fibres formed new neural lobules. This regenerative process was accompanied by an increase in blood supply from the primary plexus of the median eminence to the neuro-intermediate lobe.Supported by grants from the Swedish Natural Science Research Council and the Royal Physiographic Society of LundThe authors are indebted to Mrs. Ingrid Hallberg and Mrs. Kirsten Thörneby for valuable technical assistance and skillful attention to the animals and to Miss Inger Norling for photographic aid     

Control of the pars intermedia of the lizard,Anolis carolinensis

Summary The ultrastructure of the neural lobe of the lizard, Anolis carolinensis, was studied after fixation in a threefold aldehyde solution. The neural lobe appeared as narrow vertical diverticula separated from one another and from the pars intermedia by a continuous vascular septum. No nerves passed through this septum. The ependymal, fibrous and external layers were readily recognized. Peptidergic fibres were the main component of the fibrous layer. The peptidergic endings were in intimate contact with the ependymal cells, suggesting that the ependyma mediates the release of neural lobe peptides. The external layer contained ependymal end-feet and numerous aminergic terminals, ending directly on the perivascular basal lamina and/or on the ependymal end-feet. The functional aspects are discussed in terms of intermediate lobe control. The findings suggest that aminergic substances take part in the control of the intermedia, but do not exclude the involvement of peptide hormones.Supported by grants from the Swedish Natural Science Research Council and the Royal Physiographic Society of LundThe authors are indebted to Mrs. Lena Sandell for valuable technical assistance and to Miss Inger Norling and the late Mr. Lajos Erdös for photographic aid     

Immunohistochemical demonstration of alpha-1-antitrypsin in the islet cells of human pancreas

Summary Using the unlabeled antibody peroxidase-antiperoxidase complex (PAP) technique at the light microscopic level, it has been shown that, in the dipnoan preoptico-hypophysial neurosecretory system, vasotocin and mesotocin are synthesized in separate neurons. In the preoptic nuclei, the perikarya of these two types of neurosecretory neurons are not located preferentially. The two types of neurosecretory perikarya give rise to separate vasotocinergic and mesotocinergic axons, respectively. The dipnoan median eminence and neural lobe contain separate vasotocinergic and mesotocinergic nerve fibres, the general distribution of which is described. In the pars distalis and the pars intermedia of the hypophysis, neurohypophysial hormone-containing nerve fibres have not been found.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk OnderzoekThe authors are greatly indebted to Prof. Dr. Hyder, Department of Zoology, University of Nairobi, Kenya for kindly supplying us with the fixed material used in this study     

Vascular Connections in the Pituitary of Anolis carolinensis with Special Reference to the Pars intermedia

The pituitary vascular system of Anolis carolinensis is similar to that of other lizards. Distinct portal vessels connect the primary plexus of the median eminence with the secondary plexus of the pars distalis. The primary plexus has some connections with the neural lobe. The neural lobe plexus is fed by separate arteries, and drains into the pars intermedia. The latter lobe receives its blood supply by this route. The pituitary is drained into the vena retrohypophysea. The findings are discussed in relation to existing theories regarding the neurovascular control of the pars intermedia.     

Immunocytochemical localization of vimentin in the posterior lobe of the cat, rabbit and rat pituitary glands

The presence and distribution of vimentin, a subunit of intermediate filaments, in the neural lobe and in the pars intermedia of the cat, rabbit and rat pituitary glands were investigated immunocytochemically. In the pars intermedia, our study revealed the presence of vimentin in glial-like cells located between glandular secretory cells of the three species and in the cells of the marginal layer of the cat and rat hypophyseal cleft. In the neural lobe of the cat and rabbit pituitary glands, there was a large amount of cell processes and immunoreactive pituicytes. In contrast, in the rat neural lobe, few pituicytes exhibited immunoreactivity, and these were located principally in the posterior region near the pituitary stalk. The significance of immunoreactive vimentin in these cells is discussed.     

The hypothalamo-hypophysial system of the wild carp,Cyprinus carpio L.

Summary The posterior neurohypophysis (PNH)-pars intermedia complex of the wild and pond carp, Cyprinus carpio L., has been studied by light, fluorescence and electron microscopy. Gomori-positive neurosecretory fibres are abundant in the main trunk of the neurohypophysis as well as its roots penetrating the pars intermedia. Terminals of these fibres are in contact with capillaries of the general circulation and with glandular cells of the pars intermedia. Monoaminergic fibres with a weak green fluorescence, somewhat increasing after injection of nialamide into the pond carp, have largely the same distribution. Three types of neurosecretory fibres and their terminals have been recognized in the PNH-pars intermedia complex. Types-A₁ and -A₂ fibres, containing granules of 140–180 nm and 100–160 nm in diameter respectively, are peptidergic Gomori-positive. Type-A₂ fibres predominate in the PNH. The least frequent monoaminergic type-B fibres have granules of 60–100 nm in diameter. Numerous peptidergic and few monoaminergic neurosecretory terminals make contact with the capillaries located within the roots of the PNH as well as at the border between them and the pars intermedia. Both peptidergic and monoaminergic terminals make direct synaptoid contacts with the gland cells or end close to connective tissue septa, basal lamina or pituicytes. The PAS-positive gland cells and to a lesser degree the leadhaematoxylin-positive gland cells show these relationships with neurosecretory terminals. The question concerning the mode of interaction between peptidergic and monoaminergic structures in the dual control of the gland cells of the pars intermedia of teleosts is discussed.     

Oxytocin-immunoreactive nerve fibers in the pars intermedia of the pituitary in the rabbit and hare

The pars intermedia of the pituitary in the rabbit and hare is abundantly innervated by axons reacting selectively with antibodies against oxytocin. These axons contain dense secretory vesicles about 140 nm in diameter, i.e., smaller than those in the neurosecretory axons of the neural lobe. No fiber elements staining for other peptides (vasopressin, somatostatin, substance P) were observed in the pars intermedia, except rare leu-enkephalin axons restricted to the rostral zone of the gland. Dopaminergic innervation appears to be completely absent from the intermediate lobe. This was shown by the lack of reaction with an antibody against tyrosine-hydroxylase, which did reveal a well-developed tubero-infundibular system of nerve fibers. Axons reacting with an antibody against serotonin were irregularly distributed in the pars intermedia. In the absence of dopaminergic axons, the extensive oxytocin-like innervation may play a major role in regulating the melanotrophic cell activity in the Leporidae.     

Electron microscopical study on the development of the nerve supply of the pituitary pars intermedia of the mouse

Summary The development of the nerve supply of the pituitary pars intermedia (PI) of C3H mice was studied by electron microscopy. Nerve fibres and terminal structures, most probably adrenergic, first appear in the newborn. The adult innervation pattern is achieved by the end of the first postnatal week.In the adult animal two types of nerve terminals were distinguished; type A (peptidergic or neurosecretory) and type B (adrenergic). The peptidergic fibres were scarce and exhibited no synapse-like contacts. It is suggested that they are of secondary importance in a direct nervous hypothalamic control of PI function. Type B terminals were found throughout the PI. They formed synapse-like contacts with the glandular cells, indicating that the primary innervation is exerted by adrenergic neurons.An autonomous differentiation of the glandular cells and in the adult a combined direct nervous and neurohumoral control of PI function is suggested.This investigation was supported by grant No B 2180-026 from the Swedish Natural Science Research Council. The skilful technical assistance of Mrs Ulla Wennerberg is gratefully acknowledged     

Assessment of TRH as a potential MSH release stimulating factor in Xenopus laevis

This study considers the possible involvement of the tripeptide TRH (thyrotropin releasing hormone) in the physiological regulation of melanophore stimulating hormone (MSH) secretion from the pars intermedia of the toad, Xenopus laevis. TRH was shown to stimulate release of MSH from superfused neurointermediate lobes obtained from white-background adapted animals, but had no effect on secretion from lobes of black-background adapted animals. Immunohistochemical analysis revealed a rich TRH-containing neuronal network terminating in the neural lobe of the Xenopus pituitary. Plasma levels of TRH, determined with a specific radioimmunoassay, proved to be extremely high and no significant difference in this level could be found between white- and black-adapted animals. Plasma TRH probably originates from the skin, and our results show that its concentration is within the effective concentration range established for this peptide in stimulating MSH release from the pars intermedia. Therefore, while both our superfusion and immunohistochemical results argue favourably for a function of TRH in the regulation of MSH secretion, we conclude that, in any regulatory role, it would likely have to function within the pars intermedia at concentrations exceeding the high plasma values. While TRH could be involved in short-term activation of the secretory process in white-background adapted animals or in animals undergoing the initial stages of black background adaptation, our results indicate that this peptide may have no function in the maintenance of secretion from the pars intermedia of animals fully adapted to black background.     

Immunoreactivity to gonadotropin-releasing hormone and gonadotropic hormone in the brain and pituitary of the rainbow trout Salmo gairdneri

Summary Immunoreactivity to gonadotropin-releasing hormone (GnRH) and gonadotropic hormone (GTH) was studied at the light-microscopical level in the brain and pituitary of rainbow trout at different stages of the first reproductive cycle using antisera against synthetic mammalian GnRH and salmon GTH. GnRH perikarya were localized exclusively in the preoptic nucleus, both in the pars parvicellularis and the pars magnocellularis. A few somata contacted the cerebrospinal fluid. Not all neurosecretory cells were GnRH-positive, indicating at least a bifunctionality of the preoptic nucleus. We recorded no differences between sexes or stages of gonadal development in the location of GnRH perikarya, whereas gradual changes were found in staining intensity during the reproductive cycle. GnRH fibres ran from the partes parvicellularis and magnocellularis through the hypothalamus and merged into a common tract at the transverse commissure before entering the pituitary. In the pituitary, GnRH was localized in the neural tissue of the neurointermediate lobe and, to a lesser extent, in the neural protrusions penetrating the proximal pars distalis. The bulk of GTH-positive cells was situated in the proximal pars distalis. Some cells were found more rostrally amidst prolactin cells or in the neurointermediate lobe. Only a limited number of GTH cells appeared to be in close contact with GnRH-positive material.