Ultrastructure of the neurosecretory cells of the anterior commissural nucleus of the hypothalamus in rats

The ultrastructure of neurosecretory cells of the anterior commissural nucleus of rat hypothalamus is similar to that of the supraoptic nucleus and of the "magnocellular" part of the paraventricular nucleus. The only difference is a less expressed granular endoplasmatic reticulum and a smaller diameter of elementary neurosecretory granules (80-150 nm in diameter). Such elementary granules are characteristic of neurosecretory terminals located in the external zone of the median eminence. It is suggested that neurosecretory cells of the anterior commissural nucleus project to this neurohemal region.     

Immunohistochemical and electron microscopic study of the rat hypothalamic nuclei and cell clusters under various experimental conditions

Summary In untreated, pregnant and thirsting rats the neurosecretory hypothalamic areas were investigated by means of the immunoperoxidase technique in order to demonstrate vasopressin- and oxytocin containing elements at the light- and electron microscopic level. In addition, chromalum-hematoxylinphloxin (CHP) staining and conventional double staining of ultrathin sections were used. The areas investigated included the anterior and posterior supraoptic nuclei, the paraventricular nuclei, the numerous accessory cell clusters in the region between the tractus opticus and the third ventricle as well as the median eminence. In all nuclei and in the accessory cell clusters, the number of vasopressin-reactive neurons exceeds that of oxytocin-reactive neurons. Compared with the anterior supraoptic nucleus, the posterior supraoptic nucleus and the accessory cell clusters react more heavily to prolonged thirst. In the median eminence the neurosecretory axons display close contacts with the portal vessels not only in its lateral portion but in thirsting animals also around the mid-line. There the internal layer is broadened and vasopressin-positive tanycytic processes reach the external zone. Parasagittally, fine vasopressin-positive material can be traced from the internal layer to small deposits at the portal vessels. In long term thirsting animals the typical feature of swollen axons exhibits a characteristic distribution in the median eminence and renders a distinct positive reaction to anti-vasopressin. The release of peptide hormones from the perikarya and from the axons within the nuclei as well as the mode of release within the median eminence are discussed. The significance of the positive immunostaining of the ependymal tanycytes and of some perikarya of the suprachiasmatic nucleus must be reconsidered by further studies.Supported by the Deutsche Forschungsgemeinschaft (Grant Nr. Kr 569/1) and Stiftung VolkswagenwerkDedicated to Professor Berta Scharrer on the occasion of her 70th birthdayThe author wishes to express her special gratitude to Dr. L.A. Sternberger for supplying the peroxidaseantiper oxidase-complex and to Dr. H. Stein (Pathologisches Institut der Universität Kiel) for supplying Anti-IgG. The skilful technical assistance of Mrs. H. Prien and Mrs. H. Schöning is thankfully acknowledged     

Electron microscopic immunocytochemical investigation on the postnatal development of the vasopressin system in the rat

Summary The present ultrastructural results indicate that, in the rat, the vasopressin-synthesizing perikarya of the supraoptic nucleus (NSO) attain a certain degree of maturity earlier than those of the paraventricular nucleus (NPV). In the neonate rat, the stainability of the nuclear areas is very weak; in the perikarya of the NSO a few labeled granules can be found, whereas the perikarya of the NPV often display only a labeled Golgi area, the cytoplasm being devoid of granules. At the end of the first (NSO) and the second (NPV) postnatal weeks, the filling of the neurosecretory granules with vasopressin is inhomogeneous with irregular spots of reaction product distributed on the granules. This feature is less obvious during the following week and has nearly disappeared after the third and fourth postnatal weeks. Already in the neonate two types of vasopressin-positive fibers are observed in the median eminence, characterized by the different diameters of their granules and by their typical location in the internal and the external pericapillary contact zone. Especially in one and two week-old animals, in the internal zone of the median eminence and, to a lesser degree in the neural lobe, the immunocytochemical reaction product is deposited on an axonal tubular network. Judging from the presence of very few vasopressin-negative fibers in the neural lobe of the neonate, the development of the oxytocin system appears to be delayed. A characteristic relationship between pituicytes and the neurosecretory fibers can be observed during the first two postnatal weeks. After the third postnatal week the immunocytochemical features of the vasopressin system correspond approximately to that in adult rats.Supported by the Deutsche Forschungsgemeinschaft (Grant Nr. Kr 569/3) and Stiftung Volkswagenwerk     

Different populations of granules and their distribution in the hypothalamo-neurohypophysial tract of the rat under various experimental conditions

In connection with previous studies (Krisch, 1974), a morphologic analysis of the median eminence of male, female, pregnant, and thirsting rats has been made, in order to evaluate: 1. whether the median eminence is an additional area where oxytocin and vasopressin may be released from neurosecretory axons in the zona interna; 2. whether it is possible to distinguish different populations of axons according to the mean diameters of their granules in the external zone of the median eminence. In the zona interna of the male and the female control animals, the oxytocin- and vasopressin-containing granules have already reached their final diameters, i.e. their size remains constant until they reach and are stored in the neurohypophysis. However, the relative proportions of these populations in the fibre layer of the median eminence indicate certain differences between male and female animals, concerning the turnover of oxytocin vasopressin. In pregnant, as well as in thirsting animals, there is an apparent decrease of the mean granule diameters in oxytocin- and vasopressin-containing axons. In particular in the latter group, ballooned axons in the fibre layer of the median eminence, containing disintegrating granules, tubular structures filled with electron dense material, and fine-flocculent material in the axoplasm, together with the morphometric results, support our concept that in case of highly increased peripheral demand the contents of the mature, large granules are released into the axoplasm of the fibre layer in the median eminence. In addition, axons containing granules smaller than 115 nm may be classified into four populations with mean diameters of 84 nm, 96 nm, 103 nm, and 100n. The same populations are found in the palisade zone of the median eminence as well as in the pericapillary zone, but in contrast to the zona interna there is a fifth population of axons containing very small granules with a mean diameter of about 70-75 nm. The relative proportions of these five populations differ between the zona palisadica and the pericapillary zone on the one hand and between the experimental groups on the other hand. Some morphological findings concerning the tanycytes are discussed with respect to previously published data.     

Immunohistochemical localization of vasoactive intestinal polypeptide(VIP)-containing neurons in the hypothalamus of the Japanese quail,Coturnix coturnix

Summary The localization of vasoactive intestinal polypeptide (VIP) in the hypothalamus of the quail has been studied by means of light- and electron-microscopic immunohistochemistry. Numerous VIP-immunoreactive perikarya are distributed in the caudal portion of the nucleus infundibularis (n. tuberis) and nucleus mamillaris lateralis, and sparse in the preoptic area, nucleus supraopticus and nucleus paraventricularis. Dense localization of immunoreactive-VIP fibers is observed in the external layer of the median eminence, in close contact with the primary portal capillaries. The main origins of these fiber terminals are VIP-immunoreactive perikarya of the nucleus infundibularis. These neurons are spindle or bipolar and extend one process to the ventricular surface and another to the external layer of median eminence. They are CSF-contacting neurons and apparently constitute the tubero-hypophysial tract that links the third ventricle and the hypophysial portal circulation. VIP-reactive neurons in the nucleus mamillaris lateralis also project axons to the external layer of the median eminence, constituting the posterior bundle of the tuberohypophysial tract. Numerous VIP-immunoreactive perikarya occur also in the nucleus accumbens/pars posterior close to the lateral ventricle. They are also CSF-contacting neurons extending a process to the lateral ventricle. There are moderate distributions of VIP-reactive fibers in the area ventralis and in the area septalis.Ultrastructurally, the immunoreactive products against VIP are found in the elementary granules, 75–115 nm in diameter, within the nerve fibers in the median eminence.This investigation was supported by Scientific Research Grants No. 00556196, No. 56360027 and No. 56760183 from the Ministry of Education of Japan to Professor Mikami and Mr. Yamada     

Stress and Colchicine do not Induce the Release of Galanin from the External Zone of the Median Eminence

The aim of the present study was to verify the hypothesis that stress exposure modifies the content and release of galanin in the hypothalamic paraventricular nucleus and the median eminence. Colchicine and immobilization served as stress stimuli, and the changes in galanin immunoreactivity were compared with those in corticotropin-releasing hormone and vasopressin. In control animals, a limited number of galanin perikarya were identified in the paraventricular nucleus. The high dose (75 g) of colchicine enhanced galanin in both parvicellular and magnocellular subdivisions, as analysed 72 h later. In the median eminence, galanin accumulated only in the external zone. High- dose colchicine did not affect galanin, while corticotropin- releasing hormone and vasopressin were depleted from the median eminence. Immobilization (120 min) neither alone nor in combination with colchicine influenced galanin immuno-reactivity in the external zone. The low dose of colchicine induced an unexpected accumulation of galanin in the internal zone of the median eminence, which was further increased by subsequent immobilization. In the external zone, low-dose colchicine induced a complete disappearance of vasopressin, substantial depletion of corticotropin-releasing hormone and no changes in galanin immunoreactivity. The present studies demonstrate that galanin in the external zone of the median eminence is not influenced by colchicine or by immobilization stress.     

Ultrastructure of the peptidergic and monoaminergic neurons in the hypothalamic neurosecretory system of Anuran Batracians

Summary In the toad Bufo arenarum Hensel the following regions of the hypothalamic — neurohypophyseal system were studied under the electronmicroscope: preoptic and paraventricular nuclei, median eminence and infundibular process of the neurohypophysis.Neuronal perikarya of the preoptic nucleus are loaded with typical neurosecretory granules of peptidergic nature having a mean diameter of 1660 Å. While most neurons of the winter toad are in a storage stage a few show signs of a more active synthetic activity. A distinctive feature of preoptic neurons is the presence of large lipid droplets. The paraventricular nucleus contains small neurons containing granulated vesicles with a mean diameter of 800-1000 Å. In the region extending between these two nuclei and the median eminence axons containing either neurosecretory elementary granules or granulated vesicles are observed.The inner zone of the median eminence is occupied by axons of the preoptic neurohypophyseal tract; two types of axons, according to the size and density of the neurosecretory granules, may be recognized. The outer zone of the median eminence contains mainly axons and nerve terminals containing granulated vesicles of probable monoaminergic nature and only a few with granules of peptidergic type.The neurohypophysis contains two kinds of axons: one with more dense granules of 1800 Å and the other with granules of lesser electron density and 2100 Å. At the ending proper small clear vesicles of synaptic type are found.A progressive increase in volume of the peptidergic granules along the axon is demonstrated. This is of the order of 218% from the preoptic perikarya down to the infundibular process. The physiological significance of the two neurosecretory systems — i.e. the monoaminergic and the peptidergic — and the probable nature of the two types of peptidergic axons is discussed.Supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas and by the Air Force Office of Scientific Research (AF-AFOSR 963-67).The authors want to express their gratitude to Mrs. Defilippi-Novoa and Mr. Alberto Sáenz for their skillful assistance.     

Ultrastructural characteristics of immunolabelled,corticotropin releasing factor (CRF)-synthesizing neurons in the rat brain

Summary The corticotropin releasing factor (CRF)-synthesizing perikarya and neural processes were detected at ultrastructural level in the hypothalamic paraventricular nucleus and in the median eminence of control and colchicine-pretreated rats. The unlabelled antibody peroxidase-antiperoxidase complex (PAP) immunohistochemical method was used in a pre-embedding manner, on thick, non-frozen sections. In CRF-perikarya, neurosecretory granules (80–120 nm in diameter), free ribosomes, and the rough endoplasmic reticulum were labelled. Unlabelled axon terminals formed asymmetric synapses on CRF-containing perikarya and dendrites. Immunolabelled axons terminated in the palisadic zone of the median eminence.     

Ultrastructural immunohistochemical localization of vasopressin in the hypothalamic-neurohypophysial system of three murids

Vasopressin was immunohistochemically localized at the electron microscopic (EM) level in the hypothalamic-neurohypophysial system (HNS) of three murids. Antiserum to vasopressin was produced in rabbits injected with lysine vasopressin (LVP) conjugated to egg albumin (EA), anti-EA being precipitated prior to staining. Sternberger's unlabeled antibody peroxidase technique was employed, immunoreactivity being designated by peroxidase anti-peroxidase (PAP) molecules and electron opacity. Immunoreactive neurosecretory granules (NSG) were found in the perikarya of the supraoptic nucleus (SON) in all three murids investigated, although far more profusely in the two wild strains. Immunoreactive axonal NSG were observed in the inner and outer zones of the median eminence (ME), and within most of the axons and terminals in the neurohypophysis. The concentration of primary serum effective for staining the SON (110–150) was far higher than that required for the ME and the neurohypophysis (1:500–1:1,200). AntiLVP also induced electron opacity of granules in cells of the pars intermedia (PI). Discussion centers on the significance of immunoreactive NSG in the neurosecretory (NS) perikarya, on the possibility of an extragranular pool of hormone, and on speculation about the electron opacity of the PI granules.     

Fine structure of the median eminence and the pars nervosa of the bullfrog,Rana catesbeiana

Summary The hypothalamic neurosecretory system of the bullfrog, Rana catesbeiana, was studied with light- and electron microscopy. The median eminence is roughly divided into two portions. The upper portion mostly consists of ependymal cells, glial cells and preoptico-hypophysial nerve tract, whereas in the lower portion, neurosecretory axons, glial cells, processes of glial and ependymal cells, and fine blood vessels of the hypothalamic portal vein are located. A part of the neurosecretory axons of the preoptico-hypophysial tract proceeds to the lower portion of the median eminence. These axons are arranged perpendicularly to the capillaries of the hypothalamic portal vein. The glial cells are densely located in the area of the median eminence where neurosecretory material is abundant. The neurosecretory material in the neurosecretory cells, their axons, the median eminence and the pars nervosa of the bullfrog shows a positive reaction to PAS treatment.The neurohemal area of the median eminence is occupied by many neurosecretory and non-neurosecretory axons, containing neurosecretory granules and/or synaptic vesicles. The axonal portions with the synaptic vesicles which are considered to be the nerve endings abut on the capillaries of the portal system. The size of synaptic vesicles in the axon terminals containing few neurosecretory granules is larger than those in the endings with many neurosecretory granules. Infrequently glial and ependymal processes are interposed between the nerve endings and the capillary wall.In the hilar region of the infundibulum, synapses are frequently observed between the thin fibers with or without neurosecretory granules and dendrites of non-neurosecretory neurons. The probable functions of these synapses are briefly discussed on the basis of our findings. Both in the hilar region of the infundibulum and in the pars nervosa, electron-dense neurosecretory granules of two different sizes were observed. The median eminence contains only one type of granules.The fine structure of the pars nervosa shows similar structures to those of the median eminence. Both in the median eminence and the pars nervosa, the fenestrated endothelium of the capillaries was frequently observed. The thick perivascular connective tissue space containing fibroblasts and collagen fibrils was observed both in the median eminence and the pars nervosa. Vesicles in the cytoplasm of the endothelial cells which appear to take a part in the transendothelial transport were observed.This investigation was supported in part by United States Public Health Service Research Grant, No. A-3678, to Hideshi Kobayashi from the National Institute of Arthritis and Metabolic Diseases and partly by a grant for Fundamental Scientific Research from the Ministry of Education of Japan. The authors wish to express their thanks to Prof. K. Takewaki for his kind encouragement.     

Ultrastructural study of neurovascular contacts in the median eminence of the rat

Summary In the median eminence of the rat axons of the supraoptic-paraventricular-hypophyseal tract with elementary neurosecretory granules (150–200 m) traverse the internal zone. Terminals containing dense core vesicles 60–120 m in diameter end on the portal capillaries of the median eminence. A unique organisation of the primary portal capillaries is shown. Endothelial cells have many fenestrae. The pericapillary space has numerous extensions all of which represent a special zone around the endothelial tube. The fine structural organisation and function of the neurovascular contacts in the median eminence are discussed.     

Ultrastructural alterations of the paraventriculo-infundibular Corticotropin Releasing Factor (CRF)-immunoreactive neuronal system in long term adrenalectomized rats

The corticotropin releasing factor (CRF)-immunoreactive paraventriculo-infundibular neuronal system of long-term adrenalectomized and adrenalectomized-short term dexamethasone treated rats was analyzed at the ultrastructural level using the preembedding peroxidase anti-peroxidase complex (PAP)-immunohistological method. In both groups of animals, parvocellular neurons located in the medial and dorsal subnuclei of the paraventricular nucleus (PVN) showed CRF-like immunoreactivity. The perikarya contained hypertrophied rough endoplasmic reticulum (rER) with dilated cisternae, active Golgi-complexes and numerous neurosecretory granules. The majority of the neurosecretory granules measured 80–120 nm. Dendrites of CRF-immunoreactive neurons contained labeled vesicles, secretory granules, bundles of microtubules, a well-developed smooth endoplasmic reticulum (sER) complex and free ribosomes. Unlabeled terminal boutons of axons were observed to synapse on dendrites and somata of CRF-neurons. In addition, CRF perikarya were found in direct somato-somatic apposition with both CRF-immunopositive and immunonegative parvocellular cells. Retraction of glial processes and the existence of puncta adherentia between the cell membranes characterized these appositions. Varicose CRF axons within the median eminence contained hypertrophied sER, labeled vesicles and neurosecretory granules. The preterminal portions of the CRF-axons were dilated and possessed many labeled 80–120 nm diameter granules. CRF-terminals were greatly enlarged and established direct neurohemal contacts with the external limiting basal lamina of portal vessels without the interposition of tanycytic ependymal foot-processes. These tanycytes were not CRF immunopositive. CRF positive terminals contained clusters of microvesicles, labeled small vesicles and multivesicular bodies, but fewer granular elements than were observed within the preterminals. Many of the labeled organelles were attached to tubules of sER. Occasionally, CRF-axons were observed within the pericapillary space adjacent to portal vessels. The ultrastructural features of CRF-neurons, obtained from adrenalectomized and adrenalectomized plus short-term dexamethasone treated rats did not differ significantly from each other. The hormone content of the entire CRF-neuron was greater in the steroid treated group. Adrenocorticotrophic hormone (ACTH) synthesizing cells in the pars distalis of adrenalectomized-dexamethasone treated rats also showed increased numbers of immunopositive secretory granules (150–320 nm in diameter). These ultrastructural morphological results provide evidence that the function of the paraventriculo-infundibular CRF-system is adrenal steroid hormone dependent and suggest the participation of glial and ependymal elements in the regulation of the system in this hyperfunctional state. The observed membrane specializations are indicative of ephaptic interactions between CRF-neurons and may serve a synchronizing function in adrenalectomized animals.     

The effect of castration upon the ultrastructure of the rat hypothalamus

Summary The neurosecretory hypothalamic nuclei and the inner zone of the median eminence of castrated rats were studied under the electron microscope. After one month of castration all the neurosecretory neurons of both nuclei show signs of hyperactivity characterized by dilated cisternae of the endoplasmic reticulum containing a macromolecular filamentous material and an increase in the number of ribosomes. After six months of castration, some neurosecretory neurons show an increased number of neurotubules and larger lysosomes than in the controls. Other neurons show a very significant hypertrophy of the endoplasmic reticulum, with large amounts of intracisternal filamentous material. These cells have few neurosecretory granules and in the adjacent synapses the number of granulated vesicles is increased. In the supraoptic nucleus there are two kinds of neurosecretory axons: the clear ones, which are similar to those that appear in control animals and the dark ones, which have smaller elementary granules. In the inner zone of the median eminence the axons show an increase in the number of neurosecretory granules with respect to the controls. After supplementary administration of sexual hormones, all the modifications produced by castration disappear. The ultrastructural changes observed in the neurosecretory nuclei after castration are discussed in relation to those previously described in the neurohypophysis under the same experimental conditions. A feedback regulatory action of sex hormones on hypothalamic neurosecretory neurons is postulated.Supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas and by the Air Force Office of Scientific Research (AF-AFOSR 963-67).We are deeply indebted to Mrs. Defilippi-Novoa and Mr. Alberto Saenz for their skillful assistence.     

Fetal hypothalamic transplants in the third ventricle of the adult rat brain

Summary Blocks of anterior hypothalamus were transplanted from 19 day-old fetuses of Wistar/Lewis rats into the third ventricle of adult male Brattleboro rats. Physiological changes in graft recipients and in sham-operated animals were monitored daily. Twenty days after surgery, the graft recipients and shamoperated animals were killed and their brains examined by correlative scanning and transmission electron microscopy. Host animals that exhibited both decreased polydipsia and increased urine concentration were found to have viable grafts within the third ventricle. The observed physiological changes suggested that synthesis and release of vasopressin occurred in the transplanted neurons. Grafts were well vascularized by vessels arising from the host hypothalamus. Neurons, with perikarya ranging from 8 to 30 m in diameter, glial cells, and neurites were located throughout the transplants. A neurohemal contact zone, similar to that normally seen in the median eminence, could not be demonstrated in the grafts. The absence of complete glial and ependymal barriers indicates a relatively close association between cells in the transplants and the cerebrospinal fluid. A large increase in supraependymal neurons and their processes, including an eruption of neurons through the floor of the third ventricle in one animal, was observed in graft recipients but not in shamoperated animals.Supported by NIH Grants NS 15109 and NS 13717     

Nongranular vasopressin synthesis and transport in early stages of rehydration

Summary The vasopressin system of the rat was examined in the course of the first 12 h of rehydration after prolonged thirst at light and electron microscopic levels and by use of the peroxidase anti-peroxidase (PAP) method. Light microscopically, the median eminence was the only part of the system that not only displayed distinct differences between animals of different rehydration times but also showed a characteristic pattern of immunohistochemical reactivity in its rostro-caudal distribution. Ultrastructurally, in the perikarya a maximal labeling of the rough endoplasmic reticulum was observed after 2 h of rehydration, whereas an extensive labeling of the enlarged Golgi zones was attained after 4 h of resupplying water. A labeling of the intercellular clefts in the basal glial labyrinth of the supraoptic nucleus (and to a lesser degree in the subependymal neuropil adjacent to the paraventricular nucleus) was increased 30 min after the onset of drinking, as compared with water-deprived animals; it decreased slightly after 12 h of rehydration. The filling of the swollen fibers by increasing amounts of labeled axoplasmic reticulum, evident in the nuclear areas already after 30 min of water supply, begins in the median eminence after 2 h of rehydration and is fully developed after 4 and 8 h. Corresponding results hold true for the neural lobe but are somewhat delayed in comparison to the findings in the median eminence. The discussion considers (i) synthesis and transport of nongranular vasopressin within the axoplasmic reticulum, and (ii) release not only from the neural lobe but also from the nuclear areas and from the fibers of the median eminence.Supported by the Deutsche Forschungsgemeinschaft (Grant Nr. Kr 569/3) and Stiftung Volkswagenwerk     

Photoperiodic and osmotic influences on the ultrastructure of the hypothalamic neurosecretory system of the White-crowned sparrow,Zonotrichia leucophrys gambelii

Summary An attempt was made to correlate functional changes in the neurohypophysis of the White-crowned Sparrow, Zonotrichia leucophrys gambelii, with morphologic features on the light- and electron-microscope levels. The aldehyde-fuchsin-staining anterior median eminence possesses essentially the same ultrastructural features as the non-staining posterior median eminence. The axon terminals are characterized by the presence of a large number of small vesicles (approximately 400 Å in diameter) and occasional electron-dense granules. The more-or-less depleted anterior median eminence occasionally evident in the photosensitive bird showing testicular development is indistinguishable ultrastructurally from the more intensely staining median eminence generally characteristic of the photorefractory bird. In the median eminence, stainability and functional state do not seem to be correlated with changes in the type, size or number of vesicles. A slight increase in the number of granules was noted in the photorefractory bird but this was considered insufficient basis to account for differences in stainability.The pars nervosa, on the other hand, responded to osmotic stimuli (saline drinking water) by loss of stainability and decrease in numbers of elementary neurosecretory granules. Small vesicles are also present in the pars nervosa axon terminals, but are intermingled with neurosecretory granules in normal birds. Acute-osmotic birds, however, had axon terminals almost entirely occupied by small vesicles.It is to be emphasized that the pars nervosa and the median eminence are two structurally very different regions of the neurohypophysis. The basis for aldehyde-fuchsin staining in the median eminence appears to differ from that in the pars nervosa. The implications of these findings are considered in regard to hypothalamic control over gonadotropic activity in the White-crowned Sparrow.Dedicated to Professor Dr. W. Bargmann in honor of his 60th birthday.This investigation was supported by grant GB-2484 from the National Science Foundation to Professor Bern, grant GB-2819 from the National Science Foundation to Professor Mewaldt, and grant NB-01353 from the National Institutes of Health to Professor Farner. The authors wish to express their appreciation of the technical assistance of Mrs. Irene Brown, Mr. John Butchart, Sally S. Kibby, Mrs. Carol Nicoll, and Mr. John Striffler. Mrs. Emily Reid kindly prepared the histograms.     

Effect of fixation on the demonstration of neurophysin and "Gomori-positive" substances in neurosecretory granules of the rat hypothalamus.

Proteins reacting with neurophysin antibodies and "Gomori-positive" substances were demonstrated histochemically in hypothalamic neurosecretory material of normal and bilaterally adrenalectomized rats after two different fixations: a) picric acid-formalin (PAF) for 7 days at 37 degrees C; b) Bouin's fluid for 20 h at 4 degrees C. After PAF-fixation anti-neurophysin reactive neurosecretory granules are found in all parts of the supraoptico-hypophysial system and in the suprachiasmatic nucleus of normal and adrenalectomized animals. In the latter they can additionally be demonstrated in the outer layer of the median eminence. Amount and distribution of "Gomori-positive" substances correspond to those described for the immunoreactive material, except for the suprachiasmatic nucleus, in which the substances can not be detected; Following fixation in Bouin's fluid the immunohistochemical reactions are unchanged whereas the staining of "Gomori-positive" substances is remarkably impaired. The amounts of the substances demonstrable in the neural lobe are diminished and in the cells of the supraoptic and paraventricular nuclei as well as in both median eminence layers only traces of the substances are to be seen. The findings indicate that negative results in demonstrating "Gomori-positive" substances may be caused by inappropriate fixation and need to be controlled by immunohistochemical techniques.     

Quantitative radioautographic light and electron microscopic analysis of the localization of monoamines in the median eminence of the rat

Summary By means of light and electron microscopic radioautography a quantitative study of the regional distribution of catecholamines in the median eminence of the rat was carried out. One hour after intraventricular injection of ³H-dopamine the highest radioautographic reaction was recorded in the external zone, especially in the lateral palisade zone where many neurosecretory terminals are separated from the basal lamina of the portal pericapillary space by a glial cuff. This area showed the highest percentage (52%) of labelled catecholamine containing neurosecretory terminals as well as the maximal silver grain density per one terminal. In the medial palisade zone where direct neurovascular contacts with the capillary loops prevail, only 27 per cent of neurosecretory terminals were found to harbour tritiated dopamine. On the average 35 per cent of neurosecretory terminals in the median eminence of the rat contain catecholamines (both dopamine and noradrenaline). Pretreatment of animals with reserpine strongly reduced the binding of the label. Per cent of labelled neurosecretory terminals as well as grain density over terminals were decreased in both the medial and lateral palisade zones, although to a lesser degree in the latter. The site of origin of catecholamine fibers as well as the mode of catecholamine action at the level of the median eminence are discussed.This work was presented at the joint session of the Society of Anatomists, Histologists and Embryologists, the Society of Physiologists, and the Society of Neuroendocrinologists dedicated to the memory of Professor Wolfgang Bargmann, held in Leningrad, January 17, 1979It is a pleasure to express our deep gratitude to Prof. P.P. Rumyantzev and Dr. N.V. Seina of the Institute of Cytology of the USSR Academy of Sciences, Leningrad, for their valuable advice on electron microscopic radioautography     

Immunocytochemical localization of a substance in the eyestalk of the prawn,Palaemon serratus,reactive with an anti-FMRF-amide rabbit serum

Summary By use of a specific antiserum against the molluscan cardio-excitatory tetrapeptide FMRF-amide in combination with the PAP-method it was possible to obtain positive immunocytochemical reactions in several neurosecretory regions of the eyestalk of the prawn Palaemon serratus. FMRF-amide-like material was found in perikarya and nerve fibers of the medulla terminalis and in neurons in the lamina ganglionaris. The immunoreactivity observed in the glandular tissue located at the basal insertion of the eyestalk muscles must be ascribed to a non-specific reaction. The identification of immunopositive nerve fibers, ending on a nerve bundle in the medulla terminalis, and the fact that immunoreactive material was absent in the neurohemal sinus gland seem to indicate a neurotransmitter/neuromodulator function.     

Fluoreszenz- und elektronenmikroskopische Untersuchungen am Zwischenhirn-Hypophysensystem von Passer domesticus

Zusammenfassung Fluoreszierende aminerge Nervenfasern bzw. Endigungen und elektronendichte Granula mit einem Durchmesser von 500–1000 Å wurden bei Passer domesticus in den Perikaryen und im Neuropil des Nucleus infundibularis, im Neuropil des Nucleus supraopticus, im rostralen Abschnitt der neurosekretorischen Bahn (Region der Sehnervenkreuzung) und in der retikulären und Palisadenschicht der Eminentia mediana beobachtet. 2000–2500 Å große kontrastreiche Granula finden sich in den Perikaryen des Nucleus supraopticus, im Tractus supraoptico-paraventriculo-hypophyseus und im Hypophysenhinterlappen. Nach Reserpinbehandlung schwanden sowohl die gelbgrüne Fluoreszenz als auch das elektronendichte Material der 1000 Å und z. T. auch der 500–800 Å großen Bläschen. Dieses Ergebnis spricht dafür, daß beide Granulatypen Monoamine enthalten. Der Nucleus infundibularis ist nach unseren Befunden die wichtigste Quelle der etwa 1000 Å großen Granula in der Palisadenschicht der Eminentia mediana.

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Fluorescence- and electronmicroscopic studies on the hypothalamo-hypophysial system of Passer domesticus

Summary In Passer domesticus, fluorescent aminergic fibers and axon terminals and dense-core vesicles of 500 to 1000 Å diameter were found in the perikarya and the neuropile of the infundibular nucleus, in the neuropile of the supraoptic nucleus, in the rostral common neurosecretory pathway (region of the optic chiasma), in the tubero-infundibular tract, and in the reticular and palisade zones of the median eminence. 2000–2500 Å electron-dense vesicles were found in the perikarya of the supraoptic nucleus, in the proximal division of the common neurosecretory pathway and in pars nervosa of the hypophysis. After reserpine treatment, the yellow-green fluorescence as well as the dense material contained in the 1000 Å vesicles and partly also in the 500–800 Å vesicles disappeared. This suggested that the 1000 Å and the 500 Å dense-core vesicles in the hypothalamo-hypophysial system of Passer domesticus contain monoamines and that the infundibular nucleus is an important source of the 1000 Å dense-core vesicles in the palisade layer of the median eminence.

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Herrn Prof. Dr. Dr. E. Horstmann gewidmet.

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Mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft.

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Stipendiat der Alexander von Humboldt-Stiftung. (On leave from the Department of Veterinary Anatomy, University of Minnesota, St. Paul, Minnesota, U.S.A.).