Granulolysis in neurosecretory neurons of the rat supraoptico-posthypophyseal system

Ultrastructural and cytochemical observations on neurosecretory neurons of the rat supraoptico-posthypophyseal systems were made under experimental conditions which resulted in striking changes in the amount of neurosecretory granules and lysosomes. Attention was focused on granulolysis. At the onset of rehydration following a 4 days water deprivation, very active autophagy took place in neurosecretory axons of the neural lobe involving the marked increase in smooth endoplasmic reticulum, microvesicles and neurosecretory granules, although the latter were still very few due to previous depletion. When axonal transport was inhibited by colchicine at the onset of rehydration, granules accumulated in the perikarya while granule reloading of the neural lobe was delayed. However autophagy, although always active in axons, remained scarce in perikarya. Moreover, in the latter there was only slight evidence of crinophagy. Hypophysectomy also induced granule accumulation in the perikarya, although accompanied by little granulolysis. Images indicative of crinophagy as shown by acid phosphatase localization were few and exclusively restricted to perikarya, while autophagy occurred essentially in axons. Autophagy appeared to be the predominant process for granulolysis and might be considered here as an aspect of the general turnover of cell constituents, related to the sudden regression of hyperactivity-induced hyperthrophy, rather than as an expression of a specific regulation of an excess of secretory material.     

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     

Granule populations in oxytocin and abnormal perikarya of the supraoptic nucleus of homozygous Brattleboro rats: Effects of colchicine administration

Summary Magnocellular neurones in the supraoptic nucleus of the homozygous Brattleboro rat, which are unable to produce vasopressin, were investigated by immunocytochemistry to identify both the oxytocin cells and the abnormal neurones, which in normal animals would produce vasopressin. The abnormal cell profiles were significantly more rounded than those of the oxytocin cells. Both cell types showed evidence of hyperactivity, but the Golgi apparatus was more extensive in the oxytocin cells, probably as a result of the failure of the abnormal cells to produce vasopressin and its neurophysin and the resultant reduction in hormone packaging. Neurosecretory granules (NSG) 160 nm in diameter were found in the oxytocin perikarya but were absent from the abnormal cell bodies. In addition, a population of small dense granules (SDG) 100 nm in diameter was observed in both types of neurone, in numbers equal to the NSG in oxytocin cells.Injection of a low, non-lethal dose of the axonal transport inhibitor colchicine resulted in a rapid and equal accumulation of both NSG and SDG in oxytocin perikarya and of SDG in the abnormal perikarya after one day. The effects of colchicine were reversed 2–3 days after administration. The SDG, which may contain a co-transmitter or co-hormone substance, are thus produced at a similar rate to NSG, and appear to be transported from the perikarya for subsequent release at the nerve endings.     

Identification of the vasopressin producing and of the oxytocin producing neurons in the hypothalamic magnocellular neurosecretory system of the rat

Immuno-enzyme histochemical investigations showed that, in the magnocellular hypothalamo-hypophysial neurosecretory system of the rat, vasopressin and oxytocin are synthetized in separate neurons. Both the vasopressin neurons and the oxytocin neurons are present in both the supraoptic and the paraventricular nuclei in about the same number. Preferential location of the two kinds of rat neurosecretory neurons is not as obvious as in the bovine hypothalamus. Their perikarya do not show distinct morphological differences. The two kinds of neurosecretory perikarya are the origin of separate vasopressin-containing and oxytocin-containing axons respectively. In the neural lobe, the distribution of the two different types of axons is described.     

Preembedding colloidal gold immunostaining of hypothalamic neurons: light and electron microscopic localization of beta-endorphin-immunoreactive perikarya

A preembedding immunogold staining (IGS) procedure was developed to identify beta-endorphin/adrenocorticotropic hormone immunoreactive neurons at the light and electron microscopic levels. Colchicine-treated rats were perfused with Nakane's periodate-lysine-paraformaldehyde fixative. Vibratome sections were incubated in primary antisera followed by goat anti-rabbit immunoglobulin G coupled to 16 nm colloidal gold, and, in some cases, rabbit immunoglobulin G coupled to gold. The appearance to pink to light red perikarya, corresponding to colloidal gold deposition at antigenic sites, was monitored under the light microscope. Positive cell bodies in the arcuate region sometimes extended lateral to the nucleus. Only proximal portions of neuronal processes were stained. At the ultrastructural level, colloidal gold labeled the periphery of 90-110 nm dense neurosecretory granules in the perikaryal cytoplasm and a few proximal axons. Clusters of gold particles, appearing free in the neuroplasm, actually labeled secretory granules in adjacent thin sections. Granules associated with the Golgi apparatus were not stained. Colloidal gold labeling of mature beta-endorphin granules, but not progranules, in rat hypothalamic neurons was confirmed using the peroxidase-antiperoxidase technique. The results correlate well with data on the intracellular processing of pro-opiomelanocortin in pituitary cells and prepropressophysin in the paraventricular nucleus. These data demonstrate the first application of the preembedding colloidal gold staining method for the identification of intracellular antigens within the central nervous system. The IGS method provides a definitive marker for single or double labeling of nervous tissue at both the light and electron microscopic levels.     

Immunocytochemical evidence for the presence of a mutant vasopressin precursor in the supraoptic nucleus of the homozygous Brattleboro rat

Summary CP-14, a tetradecapeptide from the predicted mutant vasopressin precursor in the homozygous Brattleboro rat was detected immunocytochemically in the supraoptic nucleus of homozygous Brattleboro but not normal rats. The staining was localized to the periphery of the perikarya. CP-14 immunoreactivity was not found in the neural lobes, paraventricular nuclei, accessory nuclei or suprachiasmatic nuclei of either homozygous Brattleboro or normal rats. Vasopressin immunoreactivity was found in the neural lobe and in the perinuclear region of neurons of the supraoptic, paraventricular, suprachiasmatic and accessory nuclei of normal rats. Vasopressin immunoreactivity was also found in homozygous Brattleboro rats, mainly in the ventral part of the supraoptic nucleus: densely stained solitary cells were found amongst other faintly stained perikarya. In both cell-types the staining was mainly in the periphery of the perikarya. No vasopressin immunoreactivity was detected in the paraventricular nuclei, suprachiasmatic nuclei, accessory nuclei or neural lobe of homozygous Brattleboro rats.CP-14 and vasopressin immunoreactivities were found to be co-localized; both were present in the periphery of the same perikarya of the supraoptic nuclei of homozygous Brattleboro rats. Differential staining was found with antioxytocin serum in both normal rats and homozygous Brattleboro rats: separate neurons were stained for either oxytocin or vasopressin and CP-14. Immunoreactive oxytocin was found mainly in the perinuclear region of the neurons from the supraoptic, paraventricular and accessory nuclei.     

Immunocytochemical staining of supraoptic neurons from homozygous Brattleboro rats by use of antibodies against two domains of the mutated vasopressin precursor

Summary By use of an antibody against the 14 amino acids in the mutated vasopressin precursor (CP-14) characteristic of the homozygous Brattleboro rat, an immunohisto- and-cytochemical study was performed on the supraoptic nuclei of homozygous Brattleboro rats. At the light-microscopic level, varying numbers of perikarya per section exhibited a positive reaction. The most intense staining was observed in a patchy manner on the peripheral portions of the cytoplasm, its central portion being stained less intensely. The antiserum did not react with the supraoptic perikarya of the Wistar rat. In the homozygous Brattleboro rat, antibodies against normal vasopressin only rarely resulted in a positive immunoreaction. However, when it was observed, incubation of the subsequent section with CP-14-antiserum suggested a co-localization of both peptides in the same perikaryon. At the ultrastructural level, CP-14 immunoreactivity was demonstrated on the secretory cisternae of the Golgi apparatus, on lysosome-like bodies and on parts of the rough endoplasmic reticulum. With the use of an antibody against normal vasopressin, immunoreactivity was confined to very limited areas of the rough endoplasmic reticulum. The oxytocin immunoreactivity in supraoptic perikarya of Brattleboro rats did not differ from that in the Wistar rat, either at the light- or at the electron-microscopic levels.     

Studies on the transport of secretory granules in the magnocellular hypothalamic neurons of the rat

Intrathecal administration of 20 mug of vincristine sulphate in the rat induced in vivo the formation of paracrystalline inclusions mainly in axonal processes. This is associated with an impairment in the migration of neurosecretory granules as shown by their accumulation in the perikarya of the magnocellular neurons. The granules are intermixed with numerous dense bodies of various shape, sometimes with a fibrillar content, and probably of lysosomal origin. In addition to the impairment of the flow of neurosecretory granules, there is also a striking accumulation of mitochondria and synaptic vesicles, and an apparent proliferation of the smooth endoplasmic reticulum. In the posterior lobe, the axonal endings contain a large number of neurosecretory granules, intermingled with bodies of varying shapes and electron density. Occasionally, a dense membrane surrounding a group of elementary granules is observed, reacting positively for acid phosphatase. This suggests an attempted crinophagia.     

Ultrastructural characterisation of vasopressinergic terminals in the lateral septum of murine brains by use of monoclonal anti-neurophysins

Summary Synapses in the lateral septum of the murine brain have been investigated by ultrastructural immunocytochemistry, using monoclonal anti-neurophysins in both immunoperoxidase and immunogold techniques. In the region shown by light microscopy to be rich in vasopressinergic innervation, synaptic boutons containing 30 nm clear vesicles and occasional 100 nm dense-cored granules (granules) were stained by pre-embedding immunoperoxidase procedures with antisera to vasopressin-neurophysin, but not oxytocin-neurophysin; reaction product was diffusely distributed in the terminals. Terminals were symmetrical, and both axosomatic and axodendritic in type. Postembedding immunogold procedures by use of anti-vasopressin-neurophysin labeled only the 100 nm diameter granules in the terminals. Sodium meta-periodate treatment bleached immunoreactive granules, indicating the presence of a carbohydrate residue. The quantum of peptide packaged in the granules appears to be smaller than that in magnocellular neurones; nevertheless, the results indicate that, as in the magnocellular neurosecretory system, vasopressin and its neurophysin are packaged exclusively in granules, and that vasopressin in the septum is likely to be derived from a precursor comprising vasopressin, vasopressin-neurophysin and a glycosylated residue.     

Reserpine inhibits release of vasopressin from the neural lobe of the pituitary in dehydrated rats

Summary The effects of reserpine on the osmotically induced release of pituitary vasopressin were studied (i) by measuring the urinary excretion and the vasopressin content of the neural lobe of the pituitary, and (ii) by examining the ultrastructural morphology of axons in the neural lobe of dehydrated rats. After water deprivation for two days, control rats displayed characteristic antidiuretic response including a 75% reduction of urinary excretion and a sixfold decrease in vasopressin content of the neural lobe associated with a dramatic depletion of neurosecretory granules in corresponding axons. In contrast, when they received two daily injections of reserpine, animals dehydrated for two days showed both urinary excretion and vasopressin contents in the neural lobe that remained at levels comparable to those measured in the normally hydrated rats. Additionally, neural-lobe axons of such dehydrated, reserpine-treated rats displayed a normal amount of neurosecretory granules. These data indicate that reserpine inhibits release of vasopressin from the neural lobe and favour the concept of a facilitatory role of the catecholaminergic innervation in the control of hypothalamo-neurohypophysial vasopressin-secreting neurons.     

Electronmicroscopic immunocytochemical study on the vasopressin-containing neurons of the thirsting rat

Summary Whereas in thirsting animals the perikarya of the nucleus supraopticus are nearly empty of neurosecretory granules as evidenced by electron microscopic observation, the perikarya are heavily stained by light microscopic immunohistochemical staining. In an attempt to discover the substrate responsible for the positive immunohistochemical staining in thirsting rats, the neurons of the supraoptic nucleus of normal and long-term thirsting animals were compared by electron microscopic immunocytochemistry (indirect PAP-method). In controls all parts of the vasopressin-synthesizing neuron are filled with elementary granules which render a positive and uniform reaction after immunostaining with the indirect PAP-method. The positively reacting fibers in the external zone of the median eminence contain smaller granules than those of the tractus supraoptico-hypophyseus. Within the nucleus suprachiasmaticus, no positive reaction after immunostaining was found. In long-term thirsting animals PAP-complexes as markers of vasopressin are located over the ergastoplasm and over the few small elementary granules. The processes within the nucleus supraopticus and the ballooned axons in the internal zone of the median eminence exhibit free, i.e. non granule-bound, PAP-complexes. Findings in the nucleus suprachiasmaticus and the median eminence of thirsting animals correspond to those in controls. The neurohypophysis is almost completely devoid of PAP-labeled elementary granules.From these results it can be concluded that during thirst vasopressin synthesis is increased in the ergastoplasm and that the hormone is transported partly in a non granule-bound form. Direct contacts between neurosecretory cells and the basal lamina are found more often in thirst-stressed animals and are typical of neurohemal regions. It is discussed whether these neurohemal regions may develop transitionally under stress.Supported by the Deutsche Forschungsgemeinschaft (Grant Nr. Kr 569/1) and Stiftung Volkswagenwerk. This work was presented in part at the 72nd meeting of the Anatomische Gesellschaft, Aachen 1977     

Ultrastructural studies on the secretory cycle of the neurosecretory cells and the formation of herring bodies in the paraventricular nucleus of the rat

Summary The ultrastructure of the neurosecretory cells in the paraventricular nucleus of the normal male rat was studied by electron microscopy during various functional states. Four morphologically distinct types of neurosecretory cells were observed. It appears that they do not represent different classes of cells but different phases of secretory activity of a single cell type. The perikarya of the neurosecretory cells show a definite cycle of formation and transportation of secretory granules. We have designated the phases of this cycle as: (1) phase of synthesis, (2) phase of granule production, (3) phase of granule storage and (4) phase of granule transport. The neurosecretory granules appear to be moved in bulk into the axons, forming a large axonal swelling filled with granules as a result of one cycle in the neurosecretory process. Thus it may be postulated that a secretory cycle in the perikaryon of the neurosecretory cell seems to result in the formation of a Herring body in its axon, and that its content is then conveyed to the posterior pituitary.     

Intragranular co-storage of neuropeptide Y and arginine vasopressin in the paraventricular magnocellular neurons of the rat hypothalamus

Summary Certain populations of arginine vasopressin (AVP) neurons in the magnocellular paraventricular nucleus became immunoreactive for neuropeptide Y (NPY) when rats were treated with colchicine or monosodium glutamate (MSG). The co-storage of these peptides was examined by empooying a post-embedding electron-microscopic immunohistochemistry technique using goldlabeled antibodies to the two peptides. In colchicinetreated rats, the neuronal perikarya contained numerous secretory granules showing co-storage of the two peptides. The cells of the MSG-treated rats were characterized by having well-developed Golgi bodies with the granular structures also co-storing the two peptides, although the secretory granules in the perikarya were rather fewer than in the colchicine-treated rats. It is concluded that the destruction of the arcuate nucleus by MSG-treatment may potentiate the synthesis of NPY in AVP neurons, the synthesis of which is latent in intact animals.     

Ultrastructural studies on the localization of neurohypophysial hormones and their carrier proteins.

Neurophysin, vasopressin and oxytocin were localized in different portions of the supraopticohypophysial tract (SHT) using the unlabeled antibody enzyme technique at the ultrastructural level. In vasopressin-positive supraoptic perikarya, vasopressin and neurophysin were present in all neurosecretory granules. Within the zona interna of the median eminence, vasopressin and neurophysin were present in two populations of axons, one with granules of 1300-1500 A and one with granules of 900-1300 A. Following exposure of thin sections of median eminence to antiserum to neurophysin, reaction products were present in granules and in the extragranular cytoplasm in the axons with larger granules; in all other cases reaction product was confined to the granules. Vasopressin-positive fibers were also presented in large numbers of the zona externa of the median eminence and many terminated on the pituitary primary portal plexus. A few oxytocin fibers were present on the portal capillaries in the infundibular stalk. In the posterior pituitary all axon profiles were neurophysin positive. Neurophysin was present as both a granular and cytoplasmic pool. Vasopressin-containing axons account for 90% of the neuronal elements in the posterior pituitary and oxytocin for the remaining 10%. Findings on the subcellular distribution of these peptides are related to current theories on transport and release of neurohormones.     

Ultrastructural observation on the rat supraoptic neurons following acute operative stress

The response of the SON to various forms of stress is well documented. However, the effect of operative stress, which is a common and important clinical event requiring the mediation of vasopressin, has largely escaped attention. The present report describes the ultrastructural changes in the neurons of the caudal (retrochiasmatic) part of the SON following a deepseated linear incision on the dorsum of the rat. The observations were confirmed to the first forty-eight hours after trauma. At 24-hours post-operatively, a marked depletion of the neurosecretory granules was observed. This was associated with a proliferation of the granular and smooth endoplasmic reticulum, Golgi cisternae and ribosomes. A few of the neurosecretory granules were seen to lie in the close vicinity of the Golgi complexes. At 48-hours after trauma, these features persisted. In addition, an accumulation of neurosecretory granules was conspicuous in some axon pre-terminals. From the above findings, it is suggested that an increased demand for vasopressin during the early postoperative period is met by the supraoptic neurons by a liberation of their neurosecretory contents. An attempt at replenishment of the latter is evidenced by a proliferation in the membrane components and ribosomes. The pooling of neurosecretory granules in occasional axon pre-terminals may indicate an imbalance in the synthesis-secretion coupling of vasopressin.     

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.     

Immunoreactive galanin-like material in magnocellular hypothalamoneurohypophysial neurones of the rat

Summary Immunoreactive galanin-like material was recently shown to co-exist with vasopressin in parvocellular and magnocellular perikarya of the paraventricular nucleus in the anterior hypothalamus of the rat (Melander et al. 1986). Since this distribution pattern differed from our observation of oxytocin-associated galanin-like immunoreactivity (LI) in the neurohypophysis, we compared in series of 0.5-m thick sections the localisation of galanin-LI with the localisation of oxytocin and vasopressin/dynorphin in the hypothalamus, the median eminence and the neurohypophysis. In the oxytocin system, galanin-LI was intense in oxytocin varicosities of the neurohypophysis. Oxytocin perikarya of the hypothalamic supraoptic and paraventricular nuclei exhibited galanin-LI only after intraventricular injection of colchicine and when sections were treated with trypsin prior to application of the antibody. In the vasopressin/dynorphin system galanin-LI was intense in hypothalamic perikarya after colchicine injection and in neurohypophysial varicosities after treatment of the sections with trypsin. In these neurones, galanin-LI was absent or weak in all elements when treatments with colchicine or trypsin were omitted. Galanin-LI in the neurohypophysis was not co-localised with the numerous fine endings showing GABA-LI. These observations indicate that galanin-like material coexists with vasopressin and oxytocin in the respective magnocellular neurones, although not always in an immunoreactive form.     

Application of cryosubstitution in neurohormone- and neurotransmitter-immunocytochemistry.

Pituitaries of the African catfish, Clarias gariepinus, were prefixed in aldehyde fixatives, frozen in liquid propane and submitted to a cryosubstitution procedure. Ultrathin sections of the Lowicryl HM20-embedded tissue were treated with primary antisera raised in rabbits to gonadotropin releasing hormone (GnRH), vasopressin or gamma amino butyric acid (GABA) respectively. Binding of the primary antisera was visualized with goat anti-rabbit (GAR) labeled with gold. The general morphology of the tissue components in the cryosubstituted pituitaries matches with that obtained after routine embedding procedures. In addition, a strong labeling intensity of the neuropeptides/neurotransmitters investigated in the present study was demonstrated. Due to these qualities cryosubstitution provides optimal conditions for studying co-localization of neurosecretory products, using double-immunostaining procedures. In the pars distalis of the catfish pituitary several types of hypothalamus-derived nerve fibers are present between or synapting on the secretory cells. It is demonstrated that the two known catfish GnRHs are co-localized in the same nerve fiber and within these nerve fibers even co-exist in the same neurosecretory granules. GABA and vasopressin-immunolabeling each occurred in different nerve fibers. The present data demonstrate that cryosubstitution and low temperature-embedding results in an excellent morphological preservation compared to ultracryotomy and a better preserved immunoreactivity of small antigenic molecules in comparison to conventional fixation and embedding techniques.     

Immunocytochemical localization of the gonadotropin-releasing hormone-associated peptide portion of the LHRH precursor in the hypothalamus and extrahypothalamic regions of the rat central nervous system

Summary The gonadotropin-releasing hormone-associated peptide (GAP) of the LHRH precursor and the decapeptide LHRH were localized in the rat brain by immunocytochemistry in 12 to 18-day-old animals, by use of thick Vibratome sections and nickel intensification of the diaminobenzidinereaction product. Our results indicate that the GAP portion of the LHRH precursor is present in the same population of neurons that contain LHRH in the rat brain. An important difference observed was that the GAP antiserum, in contrast to LHRH antisera, stained several perikarya in the medial basal hypothalamus. GAP-immunoreactive perikarya were observed in the following regions: the olfactory bulb and tubercle, diagonal band of Broca, medial septum, medial preoptic and suprachiasmatic areas, anterior and lateral hypothalamus, and several regions of the hippocampus. In addition to the preoptico-terminal and the septopreoptico-infundibular pathways, we also observed GAPimmunopositive processes in several major tracts and areas of the brain, including the amygdala, stria terminalis, stria medullaris thalami, fasciculus retroflexus, stria longitudinalis medialis, periventricular plexus, periaqueductal gray of the mesencephalon and extra-cerebral regions, such as the nervus terminalis and its associated ganglion. These results confirm the specificity of previous immunocytochemical results obtained with antisera to LHRH. The presence of GAP immunoreactivity in nerve terminals of the rat brain indicates that GAP or a GAP-like peptide is located in the proper site to serve as a hypophysiotropic substance and/or as a neurotransmitter or neuromodulator.Supported by AKA No. 419427, OTKA No. 104, OKKFT 2.1.5.1 and NSF No. INT-8602688     

Immuno- and lectin-electron-microscopic investigation of the neural lobe of the hypophysis in the snake Natrix maura

Summary By the use of lectin histochemistry, and immunocytochemistry with antisera against bovine neurophysins I and II (NPs), arginine vasotocin (AVT) and mesotocin (MST), the neural lobe of the hypophysis in the snake Natrix maura was investigated at the light- and electron-microscopic levels. While paraldehyde-fuchsin stained virtually all neurosecretory endings, the periodic acid-Schiff reaction revealed only a portion of these elements. Furthermore, concanavalin-A and wheat-germ agglutinin lectins reacted with some but not all terminals. While in electron micrographs lectin-positive neurosecretory endings displayed medium-sized, pale neurosecretory granules, those from lectinnegative endings were larger and denser. The antiserum against the two NPs revealed the entire population of neurosecretory endings. The antiserum to AVT stained more numerous fiber elements than the antiserum to MST. Ultrastructurally, correlations concerning size and electron density can be found, on the one hand, between AVT-immunoreactive and lectin-positive neurosecretory granules and, on the other hand, between MST-immunoreactive and lectinnegative granules. The use of immuno-electron microscopy for the characterization of the different endings in the neural lobe and the presence of carbohydrates in some of them is discussed.This work was supported by the Directión General de Universidades e Investigación de la Junta de Andalucía (Grant BOJA 27/9/88) and the Direction General de Investigación Científica y Técnica (DGICYT Grant PB87 0710) Espaa