Immunocytochemical localization of TRH and autoradiographic determination of 3H-TRH-binding sites in the arcuate nucleus-median eminence of the rat

Immunocytochemical and autoradiographic localization of thyrotropin-releasing hormone (TRH)- and 3H-TRH-binding sites was studied in the arcuate nucleus-median eminence region of the rat. TRH-like immunoreactivity was found in dense granular vesicles (90-140 nm in diameter) in TRH-like immunoreactive nerve fibers and terminals. In the median eminence, the immunoreactive terminals were observed to be in direct contact with the perivascular basal lamina of the portal vessel and to form synaptoid contacts with tanycytes. In the arcuate nucleus, the immunoreactive terminals were often found to form axosomatic and axo-axonic, and/or axo-dendritic synapses. The uptake of tritiated TRH into the nerve fibers and terminals of the median eminence was also observed by autoradiography and the distribution and localization of silver grains in them were analyzed quantitatively by circle analysis. Thirty minutes after intraventricular infusion of 3H-TRH, radioactive labeling occurred in type-2 and 3-nerve fibers and terminals containing dense granular vesicles in the median eminence. It is therefore suggested that the neurons labeled after 3H-TRH infusion possess certain functions as physiological recognition sites or receptors for TRH.     

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 anti-LHRH and anti-ACTH activity in the "F" antiserum.

An antiserum which has previously been thought to be specific for LHRH-like immunoreactive material was shown to contain two populations of antibodies, one demonstrating anti-LHRH activity and the other anti-ACTH(1-24) activity. In rat and mouse, ACTH(1-24)-like immunoreactive substance is present in perikarya within the basal hypothalamus and in fibers in arcuate, periventricular and dorsomedial nuclei. LHRH-like immunoreactivity is present in fibers within the median eminence and arcuate nucleus, in a few fibers running along the ventral border of the hypothalamus, and in a small number of cell bodies within the medial basal hypothalamus.     

Localization of proopiomelanocortin (POMC) immunoreactive neurons in the forebrain of the pig

The distribution of proopiomelanocortin (POMC)-immunoreactive neurons was examined in the forebrains of nine sexually mature female pigs by indirect biotin-avidin horseradish peroxidase immunocytochemistry. Primary antiserum against ovine beta-endorphin (Bioflex #BF-EP-3-1) yielded positive staining of neuronal perikarya and processes. Adjacent control sections treated either with primary antiserum preabsorbed with beta-endorphin or substituted with normal rabbit serum lacked specific staining. POMC-immunoreactive cells were located in the anterior and intermediate lobe of the pituitary gland. POMC-immunoreactive perikarya were located in the arcuate nucleus and periarcuate area. The pituitary stalk/median eminence contained sparsely distributed POMC-immunoreactive fibers, which were confined to the zona interna. POMC-immunoreactive fibers were located in the arcuate nucleus and extended rostrally from the arcuate nucleus into the telencephalon coursing adjacent to the wall of the third ventricle as well as through the anterior hypothalamus, suprachiasmatic, supraoptic nuclei and preoptic areas to the nucleus accumbens, diagonal band of Broca, olfactory tubercle, bed nucleus of the stria terminalis and the ventro-lateral aspect of the septum. Caudal projections extended along the wall of the third ventricle to the level of the mammillary bodies and also coursed dorsally, passing through the periventricular, paraventricular, and dorsal medial nuclei of the hypothalamus to the midline thalamic nuclei and habenular nucleus. Lateral projections extended from the arcuate nucleus along the dorsal aspect of the optic tract and terminated in the amygdaloid complex. The distribution of POMC-immunoreactive perikarya and fibers is similar to that of the luteinizing hormone-releasing hormone (LHRH) fiber network. Therefore the opportunities exist, anatomically, for interactions between the POMC and the LHRH systems.     

Intracellular sites of proteolytic processing of pro-opiomelanocortin in melanotrophs and corticotrophs in rat pituitary.

A synthetic peptide (ST-1) corresponding to the cleavage site between ACTH and beta-lipotropic hormone moieties of murine pro-opiomelanocortin (POMC) was constructed and its polyclonal antibody was generated. This antiserum immunoprecipitated only POMC from extracts of AtT-20 cells. Moreover, an antiserum raised against porcine ACTH immunoprecipitated both ACTH[1-39] and POMC. When ultra-thin frozen sections of melanotrophs in rat pars intermedia were immunolabeled with anti-ST-1 followed by protein A-gold, gold particles indicating the presence of POMC were selectively found in the electron-dense secretory granules in the Golgi area. In addition, the immunolabeling was also observed in the cisternae of the Golgi apparatus and rough endoplasmic reticulum. In contrast, with a polyclonal antibody specific for alpha-melanocyte-stimulating hormone the gold particles were found exclusively in the electron-lucent secretory granules, with none seen in the electron-dense secretory granules. With anti-ACTH serum, gold particles were observed in the electron-dense and -lucent secretory granules. In corticotrophs in the pars distalis, many gold particles indicating the presence of POMC were observed in the Golgi and peripheral secretory granules, but the percentage of immunolabeling in the peripheral secretory granules varied from cell to cell. On the other hand, ACTH immunolabeling was found in almost all the secretory granules. This finding suggests that the processing of POMC in corticotrophs might occur in the relatively peripheral granules. These results suggest that the intracellular sites of POMC processing are somewhat different between melanotrophs and corticotrophs in the pituitary.     

Light and electron microscopic localization of ACTH and pro-opiomelanocortin-derived peptides in human developmental and neoplastic cells

Oncofetal aspects of ACTH and pro-opiomelanocortin (POMC)-derived peptides were studied immunohistochemically at the light and electron microscopic level in human fetal pituitary glands, pituitary adenomas, and small-cell carcinoma of the lung. ACTH, beta-endorphin, and gamma-MSH were localized in the same cells of both fetal and adult pituitary, as well as in the above-mentioned neoplastic tissues. However, alpha-MSH was observed only in the early fetal pituitary, its concentration decreasing with advancing gestational age. The adult pituitary contained only a few alpha-MSH-positive cells. By immunoelectron microscopy, ACTH in the adult pituitary was localized exclusively in the secretory granules. In fetal pituitary at 9 weeks' gestation, ACTH was localized in the perinuclear spaces (PNS), cisternae of rough endoplasmic reticulum (RER), Golgi saccules, and secretory granules. The staining pattern of ACTH in these organelles varied from cell to cell. In fetal pituitaries of greater gestational ages, ACTH was localized in secretory granules. The pituitary adenomas mimicked the staining characteristics of the adult pituitary, i.e., negative or only very occasional alpha-MSH staining and localization of ACTH in the secretory granules. The ectopic ACTH-producing tumors showed a staining pattern similar to that of the early fetal pituitary, i.e., positive staining for alpha-MSH and the presence of ACTH in PNS and cisternae of RER.     

Reversible ultrastructural changes of arcuate neurons after vinblastine injection into the median eminence of the rat

Summary The ultrastructural effects of vinblastine on the arcuate neurons and median eminence were studied in the rat. The animals were stereotaxically injected with solutions of 1 mM and 5 mM vinblastine into the median eminence and killed 3, 8 and 21 days after injection. Eight days after injection of 1 mM vinblastine the neurons of the arcuate nucleus showed marked changes. The Golgi complex was more distinct and considerable increases in the populations of dense bodies, granulated vesicles and coated vesicles were observed. Changes in the axo-somatic synapses and degenerating fibers in the surrounding neuropil were also characteristic of the experimental animals. The outer zone of the median eminence showed numerous degenerated nerve fibers and fibers engulfed by glial cell processes. Eight days after injection of 5 mM vinblastine arcuate neurons and median eminence showed similar changes, but quantitative differences were noted. A striking ultrastructural recovery of the arcuate neurons and axons in the outer zone of the median eminence was observed 21 days after injection of either 1 mM or 5 mM vinblastine. The results are discussed in relation to axoplasmic transport and axonal degeneration.Supported by CONICET and National University of Cuyo, Argentina.Members of the Scientific Research Career of the Consejo Nacional de Investigaciones Cientificas y Tecnicas, Argentina.     

Major role of cathepsin L for producing the peptide hormones ACTH, beta-endorphin, and alpha-MSH, illustrated by protease gene knockout and expression

The pituitary hormones adrenocorticotropic hormone (ACTH), beta-endorphin, and alpha-melanocyte stimulating hormone (alpha-MSH) are synthesized by proteolytic processing of their common proopiomelanocortin (POMC) precursor. Key findings from this study show that cathepsin L functions as a major proteolytic enzyme for the production of POMC-derived peptide hormones in secretory vesicles. Specifically, cathepsin L knock-out mice showed major decreases in ACTH, beta-endorphin, and alpha-MSH that were reduced to 23, 18, and 7% of wild-type controls (100%) in pituitary. These decreased peptide levels were accompanied by increased levels of POMC consistent with proteolysis of POMC by cathepsin L. Immunofluorescence microscopy showed colocalization of cathepsin L with beta-endorphin and alpha-MSH in the intermediate pituitary and with ACTH in the anterior pituitary. In contrast, cathepsin L was only partially colocalized with the lysosomal marker Lamp-1 in pituitary, consistent with its extralysosomal function in secretory vesicles. Expression of cathepsin L in pituitary AtT-20 cells resulted in increased ACTH and beta-endorphin in the regulated secretory pathway. Furthermore, treatment of AtT-20 cells with CLIK-148, a specific inhibitor of cathepsin L, resulted in reduced production of ACTH and accumulation of POMC. These findings demonstrate a prominent role for cathepsin L in the production of ACTH, beta-endorphin, and alpha-MSH peptide hormones in the regulated secretory pathway.     

Axons containing neuropeptide Y innervate arginine vasopressin-containing neurons in the rat paraventricular nucleus

Summary Synaptic connections between neurons immunoreactive for arginine vasopressin (AVP) and axon terminals immunoreactive for neuropeptide Y (NPY) were found in the magnocellular part of the paraventricular nucleus (PVN) in the rat hypothalamus. In pre-embedding double immunolabeling, NPY axon terminals labeled with diamin-obenzidine (DAB) reaction product established synaptic junctions on the perikarya and neuronal processes of AVP neurons labeled with silver-gold particles. Ultrastructural morphology of the neurons was more suitably preserved by a combination of pre- and post-embedding procedures. The presynaptic NPY terminals contained many small clear vesicles and a few cored vesicles, and DAB chromogen (immunoreaction product) was located on the surface of the vesicular profiles and on the core. The postsynaptic AVP neurons possessed many large secretory granules labeled with gold particles. At the synaptic junctions, small clear vesicles were accumulated at the presynaptic membrane, and the postsynaptic membrane was coated with a dense accumulation of fine electron dense particles. The perikarya also received synapses made by immuno-negative axon terminals containing many small clear vesicles and a few cored vesicles. These terminals were found more frequently than those containing NPY.     

Localization of ACTH in the human hypothalamus

Summary Using an antiserum to porcine ACTH and the unlabeled antibody peroxidase-antiperoxidase technique, we have found that ACTH is present in neuronal cell bodies located exclusively in the arcuate nucleus of the human hypothalamus. ACTH-containing fibers are distributed extensively throughout the hypothalamus with the greatest density in the periventricular nucleus. No concentration of ACTH fibers could be observed in the neurovascular zone of the pituitary stalk.     

Axons containing neuropeptide Y innervate arginine vasopressin-containing neurons in the rat paraventricular nucleus. Dual electron microscopic immunolabeling

Synaptic connections between neurons immunoreactive for arginine vasopressin (AVP) and axon terminals immunoreactive for neuropeptide Y (NPY) were found in the magnocellular part of the paraventricular nucleus (PVN) in the rat hypothalamus. In pre-embedding double immunolabeling, NPY axon terminals labeled with diaminobenzidine (DAB) reaction product established synaptic junctions on the perikarya and neuronal processes of AVP neurons labeled with silver-gold particles. Ultrastructural morphology of the neurons was more suitably preserved by a combination of pre- and post-embedding procedures. The presynaptic NPY terminals contained many small clear vesicles and a few cored vesicles, and DAB chromogen (immunoreaction product) was located on the surface of the vesicular profiles and on the core. The postsynaptic AVP neurons possessed many large secretory granules labeled with gold particles. At the synaptic junctions, small clear vesicles were accumulated at the presynaptic membrane, and the postsynaptic membrane was coated with a dense accumulation of fine electron dense particles. The perikarya also received synapses made by immuno-negative axon terminals containing many small clear vesicles and a few cored vesicles. These terminals were found more frequently than those containing NPY.     

Glucagon-related peptides in the rat hypothalamus

Summary Immunohistochemically, nerve fibers and terminals reacting with anti-N-terminal-specific but not with anti-C-terminal-specific glucagon antiserum were observed in the following rat hypothalamic regions: paraventricular nucleus, supraoptic nucleus, anterior hypothalamus, arcuate nucleus, ventromedial hypothalamic nucleus and median eminence. Few fibers and terminals were demonstrated in the lateral hypothalamic area and dorsomedial hypothalamic nucleus. Radioimmunoassay data indicated that the concentration of gut glucagon-like immunoreactivity was higher in the ventromedial nucleus than in the lateral hypothalamic area. In food-deprived conditions, this concentration increased in both these parts. This was also verified in immunostained preparations in which a marked enhancement of gut glucagon-like immunoreactivity-containing fibers and terminals was observed in many hypothalamic regions. Several immunoreactive cell bodies were found in the ventromedial and arcuate nuclei of starved rats. Both biochemical and morphological data suggest that glucagon-related peptides may act as neurotransmitters or neuromodulators in the hypothalamus and may be involved in the central regulatory mechanism related to feeding behavior and energy metabolism.     

促生长素抑制素样免疫反应在大白鼠弓状核和正中隆起的超微结构分布

本文用免疫电镜方法证明:促生长素抑制素样免疫反应神经末梢分布于弓状棱并与未标记的树突形成轴树突触。在正中隆起的纤维层和栅状层内均可见上述免疫反应末梢,大多数紧贴门脉毛细血管基底膜周围甚至穿入基底膜内。免疫反应末梢尚可与未标记的末梢形成轴轴突触样结构。     

Electron microscopic immunohistochemical localization of alpha-msh in the rat brain.

Using an immunohistochemical technique at the electron microscopic level, it has been shown that alpha-MSH is localized within the small vesicles of a few cell bodies found in the arcuate nucleus and numerous nerve fibers widely distributed throughout the brain. These findings suggest that alpha-MSH could possibly be considered as a neurotransmitter.     

Action of pro-opiomelanocortin products on the rat vas deferens

Behavioral study of pro-opiomelanocortin products indicates that beta-endorphin and corticotrophin-like peptides have antagonistic effects. However, these peptides have similar actions on the rat vas deferens. beta-endorphin, alpha-MSH and ACTH each inhibit electrically evoked contraction of the duct, but the corticotrophin derived peptides are tenfold more potent on a molar basis (ED50 = 9 nM). Pharmacological analysis shows that the action of corticotrophin-derived peptides does not involve an opiate receptor mechanism. The results are discussed in terms of the central action of the peptides.     

MSG effects on beta-endorphin and alpha-MSH in the hypothalamus and caudal medulla

Monosodium glutamate (MSG) was given to neonatal male rats to determine its effects on neurons containing beta-endorphin (beta-END) and alpha-melanocyte stimulating hormone (alpha-MSH) within the basal hypothalamus (arcuate nucleus) and caudal medulla [nucleus tractus solitarius (NTS)] and on the levels of beta-END and alpha-MSH within these areas. Immunocytochemical studies demonstrated a reduction in the number of cells within the medial hypothalamic area (arcuate nucleus) among MSG-treated animals versus saline controls. MSG did not reduce the number of cell bodies within the caudal medulla (NTS). MSG significantly reduced beta-END and alpha-MSH immunoreactive levels in the basal hypothalamus as determined by radioimmunoassay. Whereas a significant reduction in the level of beta-END occurred in the ventral caudal medulla (VCM), none occurred in the dorsal caudal medulla (DCM). In contrast, levels of alpha-MSH increased significantly in the DCM among animals receiving MSG compared to control animals. This study documents the contribution of beta-endorphin containing neurons of the basal hypothalamus to areas of the caudal medulla. The effect of MSG on beta-endorphin and alpha-MSH neurons in these areas and their differential effects on levels in the caudal medulla areas raises questions about the sites of origin of these peptides.     

Organization of tyrosine hydroxylase-immunoreactive neurons in the di-and mesencephalon of the American bullfrog (Rana catesbeiana) during metamorphosis

Summary We examined the immunocytochemical distribution of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis, in the di-and mesencephalon of developing bullfrog tadpoles. Special attention was given to catecholaminergic innervation of the median eminence and pituitary. In premetamorphic tadpoles, tyrosine hydroxylase-immunoreactive neurons were visualized in the suprachiasmatic and infundibular hypothalamus, the ventral thalamus, and midbrain tegmentum by Taylor-Kollros stage V. The number of labeled neurons in all these areas increased as metamorphosis progressed. By mid-prometamorphosis, labeled neurons appeared in the preoptic recess organ as well as in the posterior thalamic nucleus. The majority of cells in the preoptic recess organ, as well as occasional neurons in the suprachiasmatic nucleus, exhibited labeled processes which projected through the ependymal lining of the preoptic recess to contact cerebrospinal fluid. The modified CSF-contacting neurons of the nucleus of the periventricular organ were devoid of specific staining. By late prometamorphosis, labeled fibers from the suprachiasmatic nucleus were observed projecting caudally to enter the hypothalamo-hypophysial-tract en route to innervating the median eminence and pituitary. Labeled fibers arising from the dorsal infundibular nucleus projected ventrolaterally to contribute to catecholaminergic innervation of the median eminence and pituitary. Immunoperoxidase staining of tyrosine hydroxylase-immunoreactive fibers and terminal arborizations in the median eminence were restricted to non-ependymal layers, while labeled fibers in the pituitary were observed in the pars intermedia and pars nervosa. Staining of tyrosine hydroxylase-immunoreactive fibers in the median eminence and pituitary was sparse or absent in premetamorphic tadpoles, but became increasingly more intense as metamorphosis progressed.     

Immunocytochemical localization of growth hormone releasing factor (GHRF)-containing structures in the rat brain using anti-rat GHRF serum

The distribution of growth hormone releasing factor (GHRF) immunoreactive structures in the rat hypothalmus was studied after colchicine treatment with PAP immunocytochemistry in vibratome sections using an antiserum directed to rat hypothalamic GHRF. The majority of the GHRF-immunoreactive cell bodies were found in the arcuate nucleus, the medial perifornical region, and the ventral premammillary nuclei of the hypothalamus. Scattered cells were seen in the lateral basal hypothalamus, the medial and lateral portions of the ventromedial nucleus, and the dorsomedial and paraventricular nuclei. Immunoreactive fibers were observed in all the regions mentioned above. GHRF terminals were located in the central region of the median eminence. In addition, GHRF-immunoreactive neuronal processes were seen in the ventral region of the dorsomedial nucleus, the medial preoptic and suprachiasmatic regions, dorsal portion of the suprachiasmatic nucleus, bed nucleus of the stria terminals and the hypothalamic portion of the stria terminals. The localization of GHRF-immunoreactive terminals in the median eminence reinforces the view that GHRF plays a physiological role in the regulation of pituitary function. In addition, the localization of GHRF-immunoreactive structures in areas not usually considered to project to the median eminence suggest that GHRF may act as a neuromodulator or neurotransmitter.     

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     

An antibody specific for an endoproteolytic cleavage site provides evidence that pro-opiomelanocortin is packaged into secretory granules in AtT20 cells before its cleavage

We have raised a rabbit antiserum against a synthetic peptide corresponding to the cleavage site between beta-lipotropic hormone and the ACTH moieties of murine pro-opiomelanocortin (POMC). After affinity purification, the anti-cleavage site antibody immunoprecipitates POMC from extracts of AtT20 cells but it does not immunoprecipitate the ACTH in such extracts or any of the other products of cleavage of POMC. By contrast, an antiserum raised against pure swine ACTH immunoprecipitates both POMC and ACTH from AtT20 cell extracts. Using the anti-cleavage site antibody we have shown that all the POMC synthesized during a 15-min pulse-labeling with [35S]methionine is cleaved at this site within 1 h. By immunoelectron microscopy we show that approximately 25-30% of peripheral secretory granules in AtT20 cells can be labeled with the anti-cleavage site antibody while anti-ACTH antiserum labels all these granules. This establishes that at least some POMC is packaged into secretory granules before its proteolytic cleavage.