Indirect Peroxidase Research Articles

El virus de la D.V.B. como agente contaminante en cultivo de tejidos animales

The Hovine Viral Diarrhoea Virus (BWV)belongs to the genus Pestisvirus. It is the cause or reproductive disorders in catlle and is widespread in the conuntry. There are noncytophatogenic strains of the BVDV very difficult to detect inthe primary tissue culture and cell lines, ordinarilly used in diagnostic test or research activities. Foetal calf serum (FCS) is the common source of contamulation for the animal tissue culture, because of the poor quality controll during its collection and markenting under local conditions. It is possibly to fmd two kind of situations related to BVDV and the presence of viral particles; in each case is possible to have problems with the laboratory procedures. In order to avoid BWV tissue culture contamination, in our laboratory it has been used an indirect inmunofluorescense tedinique for the detectionofthe Wusinfoetalcaüserumandtissue cultures, havingresults asfoilows: 28.5%positives from 32 FCS samples from differnt sources and 42.8% from tissue cuitures from different animal origen and several research centres. Come recomdations are made for the prevention and control ot this problem at diagnostic and researchlaboratorylevelliietheuse of BI, indirect peroxidase techniques, or inmunopresitation for the detection of the Wus, and the use of a few number of fwtuses for each lot of FCS in order to reduce the risk of contamination from this source.

The distribution of fibronectin in the placental bed in normotensive and hypertensive human pregnancies

Endovascular trophoblast invasion into the spiral arteries of the placental bed is restricted to the decidual segments in pregnancy-associated hypertension (pre-eclampsia). Little is known about mechanisms that control trophoblast invasion. Extracellular matrix proteins such as fibronectin could be involved, as they are in other invasive processes. The observation of increased plasma fibronectin levels in patients that will develop pre-eclampsia (Ballegeer et al., 1989) prompted us to study the distribution of this glycoprotein in the placental bed of normotensive and hypertensive pregnant patients. Paraffin embedded sections were stained for fibronectin using the indirect peroxidase labeled antibody technique. Spiral arteries undergoing physiological changes stained virtually negative. In vessels with subintimal thickening an occasional fibronectin positive lining of endothelium and positive areas in the fibrous subintimal cushions could be found. The most intensive staining for fibronectin however was found in the arteries with acute atherosis, i.e., the vascular lesion that is thought to be associated with pre-eclampsia.

Immunohistochemical evidence for the coexistence of substance P, thyrotropin‐releasing hormone, GABA, methionin‐enkephalin, and leucin‐enkephalin in the serotonergic neurons of the caudal raphe nuclei: A dual labeling in the rat

By means of dual immunohistochemical labeling on the same brain section examined with a light microscope, the present study reports the presence with serotonin (5-hydroxytryptamine; 5-HT) of gamma-aminobutyric acid (GABA), substance P (SP), thyrotropin-releasing hormone (TRH), leucin-enkephalin (LEU-enk), or methionine-enkephalin (MET-enk), within the same neuron in the nuclei raphe magnus, raphe obscurus, and raphe pallidus of the rat. On the one hand, peptides or GABA are detected with specific rabbit antibodies by indirect peroxidase labeling using peroxidase-conjugated Fab fragments, and on the other, 5-HT is detected with a rabbit antibody against the BSA-serotonin conjugate by radio-immunocytochemistry using [125I]-labeled protein A. The possible coexistence of TRH and SP in these neurons is also investigated by using peroxidase labeling and radio-immunocytochemical detection, respectively. In the whole caudal raphe nuclei the proportion of each coexisting peptide with 5-HT appears in decreasing order as: TRH greater than SP greater than MET-enk # LEU-enk greater than GABA. In all instances the level of coexistence differs considerably in B1-B2 vs. B3 cell groups. No SP/TRH dually labeled cells have ever been found in any of the serotonergic nuclei of the caudal raphe. Given the evidence that these raphe nuclei project possibly to the spinal cord, these data constitute an anatomical substrate for the several distinct physiological functions presumably subserved by 5-HT in the cord, namely the modulation of nociception, motor, and autonomic functions.

Immunohistochemical localization of environmentally induced cytochrome P450IA1 in multiple organs of the marine teleost Stenotomus chrysops (scup)

Differences in expression of cytochrome P450 forms and their functions in different organs and cell types could determine the response of those cells and organs to xenobiotics. Recently, we described the cellular localization of cytochrome P450IA1 (P450E) induced in 10 organs or organ systems of the fish, Stenotomus chrysops (scup) treated with 3,3′,4,4′-tetrachlorobiphenyl or with 2,3,7,8-tetrachlorodibenzofuran. (R. M. Smolowitz, M. E. Hahn, and J. J. Stegeman, Drug Metab. Dispos. 19, 113, 1991). Here we describe the presence and localization of P450IA1 in organs of scup sampled directly from an environment contaminated by chlorinated biphenyls and dibenzofurans, the outer New Bedford Harbor of Massachusetts. Western blot analysis of microsomes from selected organs (liver, kidney, gill, and heart), using monoclonal antibody 1-12-3, revealed induced levels of P450IA1 in each. The localization of P450IA1 in these and other organs was determined in sections prepared by standard histological methods and stained with MAb 1-12-3 in an indirect peroxidase labeling method. P450IA1 was detected in multiple cell types in liver, including hepatic, pancreatic, and vascular tissue. Kidney and gut also showed prominent P450IA1 levels in epithelial structures and in vascular endothelial cells. Specific staining was detected in endothelial cells, but not other cell types, in heart, gill, spleen, testis, ovary, nose, and brain. In heart, the staining was present in the endocardium of atrium and ventricle, and endothelium of the coronary vasculature and great vessels. The results demonstrate that P450IA proteins are induced in many organs of fish exposed to environmental chemicals in the wild, with patterns of cellular localization like those seen in fish experimentally treated with known inducers. The strong staining of P450IA1 in endothelial cells in all organs examined supports experimental results indicating that endothelium is a major site of P450IA1 induction. Our results indicate further that immunohistochemistry is a useful method for detecting P450 induction as a biomarker for exposure.

Distribution of Immunocompetent Cells in Normal Nasal Mucosa: Comparisons among Germ-Free, Specific Pathogen-Free, and Conventional Mice

To better understand the role of immunocompetent cells in the defense mechanism of the upper respiratory tract against microbial invasions, the distribution patterns of those cells were investigated in nasal mucosa of mice maintained in three different conditions: germ-free (GF), specific pathogen-free (SPF), and conventional (CV) conditions. Immunostaining by the indirect peroxidase method and toluidine blue staining were employed for the detection of immunocompetent cells and mast cells. For immunostaining, anti-IgG, -IgA, and -IgM polyclonal antibodies and anti-Lyt-1, -Lyt-2, and -Mac-1 monoclonal antibodies were used as primary antibodies. In nasal mucosa of CV mice, Mac-1+ cells, mast cells, and all cell types of lymphocyte subsets were present. In nasal mucosa of SPF mice, all cell types were also positive, but fewer in number than those of CV mice. In nasal mucosa of GF mice, IgG+, IgA+, and Lyt-2+ cells were rare, although IgM+ and Lyt-1+ cells were present in small numbers. An electron microscopic study revealed that follicle-like lymphocyte aggregates with high endothelial venules were present in nasal mucosa close to the mucosal epithelia. These findings suggest that lymphocytes are mobilized to nasal mucosa, responding to continuous antigenic stimuli, and play an important role in the local defense mechanism of the upper respiratory tract.

The detection of human cytomegalovirus immediate early antigen in peripheral blood leucocytes

Recently, Van der Bij et al. (1988) reported that active human cytomegalovirus (HCMV) infection could be diagnosed by the detection of HCMV immediate early antigen (IEA) directly in the peripheral blood leucocytes of renal transplant recipients. However, the indirect peroxidase technique used resulted in high background staining due to endogenous peroxidase activity and thus the detection of HCMV-IEA positive leucocytes, which are sometimes present in extremely low numbers, was not always reliable. In an attempt to solve this problem, we have evaluated the alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique, immunogold-silver staining (IGSS), and several fixatives. Fixation with acetone: methanol 1:1 in conjunction with the APAAP technique proved to be the most successful method. In 155 blood samples obtained from 44 patients following renal transplantation and from three AIDS patients, the number of positive cells ranged between 1 and 700 out of 400,000 (median 2). In 23 samples from 11 patients (one AIDS patient) at least one positive cell was found. In this series there were no problems with the evaluation since strong positive signals were obtained without any background staining. We therefore recommend the use of this protocol for the rapid and reliable detection of HCMV-IEA in peripheral blood leucocytes.

Mycobacterium paratuberculosis and Crohn's disease.

The possible aetiological role of Mycobacterium paratuberculosis in Crohn's disease was investigated. The immunological response was studied using an enzyme-linked immunosorbent assay (ELISA), Western blotting, and immunocytochemistry. The antibody response to two protoplasmic antigen preparations of M paratuberculosis in the sera of patients with inflammatory bowel disease was measured by ELISA. IgG and IgM antibodies to these antigens were measured in serum samples from 52 patients with Crohn's disease, 15 patients with ulcerative colitis, and 41 control patients without inflammatory bowel disease. Although there was wide variation in the concentrations of antibody detected, patients with Crohn's disease had concentrations that were not significantly different from those of the other two groups. In addition, mycobacterial antigens were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and the immune response to each antigen was then examined separately and assayed for IgG and IgM in 10 patients from each of the three groups. An indirect peroxidase test was also used to detect M paratuberculosis in sections of tissue from 18 patients with Crohn's disease and 10 with ulcerative colitis. The results were negative in all cases. This study does not support a role for M paratuberculosis in Crohn's disease.

Immunocytochemical demonstration of the binding and internalization of growth hormone in GERL of Chang hepatoma cells

The binding and internalization of endogenous growth hormone in Chang hepatoma cells were localized on the cell surface and in the Golgi-endoplasmic reticulum-lysosome (GERL) area by various indirect immunocytochemical labeling techniques, namely, peroxidase or colloidal gold conjugated to secondary antibody, and avidin-biotin complex methods. Rabbit antiserum and monoclonal antibodies raised against HPLC-purified porcine growth hormone were used in this study. In fixed material, antigen-antibody complexes were found to be homogeneously distributed along the cell membrane. Control groups showed negative binding on the cell surface. Trypsin treatment before immunolabeling removed antibody binding completely, but hyaluronidase was ineffective. Pretreatment of lectins did not block the recognition of primary antibody to antigen molecules on cell surface. Internalization of the antigen-antibody peroxidase or gold complexes was demonstrated in the cells, which were immunolabeled at 4 degrees C, and then reincubated for 0-30 min at 37 degrees C before fixation. After reincubation, the internalized ligand complexes were found in vesicles near the cell surface or in the GERL area near the Golgi apparatus which, however, did not label for peroxidase. These findings suggest that the trypsin-sensitive growth hormone, specifically bound and internalized into Chang hepatoma cells, is localized in the GERL instead of the Golgi apparatus and might be involved in the mechanism of tumor cell growth.

Immunohistochemical demonstration of Mycoplasma gallinarum and Myoplasma gallinaceum in naturally infected hen oviducts

Using indirect immunoperoxidase (np), peroxidase anti-peroxidase (PAP) and avidin biotin-peroxidase complex (ABC) immunohistochemical methods, Myoplasma gallinarym and M gallinaceum antigens were demonstrated in ethanol-fixed paraffin-embedded oviduct sections from hens the eggs from which showed suboptimal hatchability. Specific immunoperoxidase staining was detected at the mucosa in the magnum portion of the oviduct. Optimal staining was achieved by applying the ABC method, though both nP and PAP methods can also be used for diagnosis. Isolation and identification techniques gave similar results for the species of avian mycoplasmas involved.

100 IMMUNE RESPONSE AFTER HETEROLOGOUS SURFACTANT TREATMENT OF NEWBORNS WITH RESPIRATORY DISTRESS SYNDROME (RDS)

Curosurf, a surfactant preparation derived from minced pig lungs, used for the treatment of newborn infants with RDS, contains low molecular weight proteins. We studied the immune response after a single dose of Curosurf given intratracheally in 68 patients with RDS, 49 treated with Curosurf and 19 controls. Sera were taken before, 3 wks and 3 months after surfactant treatment. The sera were applied on frozen pig lung tissue, followed by an indirect peroxidase assay. Using this procedure any immune response to lung tissue components would be demonstrated as a brown-reddish deposit. In none of the controls nor in the sera obtained before Curosurf treatment we found any reaction. In 4 of the 49 Curosurf-treated patients we found a positive immune response, demonstrated by a clear brown staining of bronchial walls and brown granular deposits in alveolar cells, and in some lung tissue specimens collagen strands were also slightly brown coloured. Of these 4 patients, 2 showed a positive reaction 3 wks and 3 months after treatment, 1 only 3 wks after and 1 only 3 months after Curosurf treatment. We conclude that sera of some patients treated with Curosurf appear to contain antibodies against pig lung tissue components and surfactant proteins.

Distribution of FMRFamide-like immunoreactivity in the brain, retina and nervus terminalis of the sockeye salmon parr, Oncorhynchus nerka

Neurons displaying FMRFamide(Phe - Met - Arg - Phe - NH2)-like immunoreactivity have recently been implicated in neural plasticity in salmon. We now extend these findings by describing the extent of the FMRF-like immunoreactive (FMRF-IR) system in the brain, retina and olfactory system of sockeye salmon parr using the indirect peroxidase anti-peroxidase technique. FMRF-IR perikarya were found in the periventricular hypothalamus, mesencephalic laminar nucleus, nucleus nervi terminalis and retina (presumed amacrine cells), and along the olfactory nerves. FMRF-IR fibers were distributed throughout the brain with highest densities in the ventral area of the telencephalon, in the medial forebrain bundle, and at the borders between layers III/IV and IV/V in the optic tectum. High densities of immunoreactive fibers were also observed in the area around the torus semicircularis, in the medial hypothalamus, median raphe, ventromedial tegmentum, and central gray. In the retina, immunopositive fibers were localized to the inner plexiform layer, but several fiber elements were also found in the outer plexiform layer. The olfactory system displayed FMRF-IR fibers in the epithelium and along the olfactory nerves. These findings differ from those reported in other species as follows: (i) FMRF-IR cells in the retina have not previously been reported in teleosts; (ii) the presence of FMRF-IR fibers in the outer plexiform layer of the retina is a new finding for any species; (iii) the occurrence of immunopositive cells in the mesencephalic laminar nucleus has to our knowledge not been demonstrated previously.

Immunohistochemical localization of androgen receptors with mono- and polyclonal antibodies to androgen receptor

Rat, human, and mouse tissues were stained immunohistochemically using mono- and polyclonal androgen receptor antibodies. Monoclonal antibodies were raised in rats and used to stain human and mouse tissues; polyclonal antibodies were raised in rabbits and used to stain rat tissues. Frozen tissue sections were incubated with the appropriate androgen receptor antibody and staining was completed by the indirect avidin-biotin peroxidase method. A comprehensive survey of rat and mouse tissues was performed. Antibody staining was found exclusively in the nucleus of certain specific cell types, suggesting that the androgen receptor is a nuclear protein. All male sexual organs in the rat showed strong positive nuclear staining for androgen receptor. Weaker positive reactions were seen in kidney, liver, adrenal cortex and pituitary gland. Furthermore, positive staining for androgen receptor was exhibited in skeletal, cardiac and smooth muscle cells, and central nervous tissue. Female reproductive organs also contained androgen receptor-positive cells. The spleen was found to be the only organ examined which did not stain for androgen receptor. The monoclonal antibody could also demonstrate androgen receptor-positive cells in a human prostatic cancer and in a prostate with benign hyperplasia. These data demonstrate the use of antibodies in revealing cellular/subcellular distribution of androgen receptor in target tissues.

Major histocompatibility complex class II antigen (HLA-DR) expression by ileal epithelial cells in patients with seronegative spondylarthropathy.

Major histocompatibility complex molecules act as non-specific receptors for antigenic proteins and present them to T-cells. Presented antigen together with class II molecules activates antigen specific T-helper cells and may trigger a cellular immune response. The expression of HLA-DR antigens by epithelial cells was examined with an indirect peroxidase technique in ileal biopsies from 38 patients with seronegative spondylarthropathy and features of acute or chronic gut inflammation on biopsy, 14 patients with chronic inflammatory bowel disease, 10 rheumatic and 10 non-rheumatic controls. In acute ileitis, there was more HLA-DR expression in villous and crypt epithelial cells than in non-inflamed controls (p less than 0.01). In chronic inflammation and in chronic inflammatory bowel disease, class II antigens were more expressed in villus (p less than 0.02) and crypt epithelium (p less than 0.01). Strong HLA-DR expression in crypt epithelial cells was connected with active inflammation (p less than 0.02). These findings suggest binding of unknown enterobacterial or nutritional luminal antigens to HLA-DR antigens normally present in enterocytes. The enterocytes act as antigen presenting cells causing a local increase of targets for activated T-cells and trigger the gut inflammation responsible for the clinical symptoms of the seronegative spondylarthropathy.

Immunocytochemical localization of vasotocin-like immunoreactivity in the brain of the cartilaginous fish, Scyliorhinus caniculus

The distribution of vasotocin-like peptides in the central nervous system of the cartilaginous fish Scyliorhinus canicula was determined by indirect immunofluorescence and peroxidase anti-peroxidase techniques, using a specific antiserum raised in rabbits against synthetic vasotocin. Immunoreactive perikarya were mainly detected in the anterior hypothalamus, within the midcaudal part of the preoptic nucleus. The most rostral positive cell bodies were located in the dorso-lateral parts of the preoptic area, whereas at a more caudal level, they took a ventro-medial position within the deepest layers of the nucleus. Throughout the preoptic region these cells varied in shape according to their location. Occasionally, scattered vasotocin-like immunopositive cells were also identified in the nucleus periventricularis hypothalami. Vasotocin immunoreactivity was detected in numerous varicose nerve fibers of the preopticohypophysial tract. These fibers were seen to course through the medio-basal hypothalamus and caudally, after having passed the hypophysial stem, they reached the neurointermediate lobe of the pituitary. Numerous immunoreactive fibers were also observed within the rostro-medial region of the median eminence. At this level the fibers were in close proximity to the capillary loops. In the preoptic region, some stained cells exibited short processes that appeared to contact non-reactive perikarya. By comparing the distribution of vasotocin- and corticotropin-releasing factor immunoreactivity on adjacent then serial sections, it was revealed that these peptides, in S. canicula, do not coexist in the same perikarya. The present results, are compared with those obtained in other vertebrate groups, and their possible functional implications are discussed.

Cell Kinetic Study on Experimental Tongue Carcinoma in Rats

Tongue carcinomas were produced in rats by oral administration of 0.001% 4-nitroquinoline 1-oxide (4NQO) in drinking water, and the biological characteristics and tumor kinetics were correlated. Bromodeoxyuridine (BrdU) was used to investigate cell kinetics of rat tongue tumors in vivo. BrdU-labeled S-phase cells were demonstrated by an indirect peroxidase method using anti-BrdU monoclonal antibody, and their percentage, the labeling index (L.I.), was determined. The average BrdU L.I. in normal tongue epithelium, well differentiated and moderately differentiated squamous cell carcinoma was 5.7 +/- 1.1, 8.8 +/- 3.7 and 14.9 +/- 5.4, respectively. The differences among the three groups are significant. These results indicate that the BrdU L.I. correlates well with the degree of differentiation of tongue epithelium. Furthermore, the distribution patterns of BrdU-labeled cells in the tissue sections of each group were different. If these cell kinetics studies using BrdU are applied to human tongue carcinomas, the data may provide information on the biological characteristics and help in establishing the diagnosis and in choosing treatment regimens.

Immunocytochemical demonstration of mineralocorticoid receptors in rat and human kidney

Using a polyclonal antiserum against the hinge region of the recently cloned human mineralocorticoid receptor (MR) and indirect peroxidase immunohistochemistry, we have shown MR-like immunoreactivity (LI) in superficial nephron segments, including distal convoluted tubule, connecting piece and initial cortical collecting duct. The absence of staining in cells tentatively identified as intercalated cells on light microscopy was confirmed by pre-embedding electron microscopy. Though the intracellular distribution of immunostaining varied with the fixative used, the cellular distribution of MR-LI is in good general agreement with earlier micropuncture and autoradiographic studies.

Ontogenesis of enkephalinergic afferent systems in the opossum cerebellum

Enkephalin (ENK) immunoreactive climbing fibers, mossy fibers and a beaded plexus of axons are present in the adult opossum's cerebellar cortex. We have used the indirect antibody peroxidase—antiperoxidase technique to study the ontogeny of enkephalinergic axons in the cerebellum of pouch young opossums from postnatal day (PD) 1 to PD 83. On PD 1, ENK axons are present in the intermediate layer of the cerebellar anlage. At PD 18, after a period of ‘waiting’, ENK fibers form clusters throughout the cerebellar cortex primarily within the nascent Purkinje cell layer. By PD 40, axon terminals with a climbing fiber phenotype circumscribe Purkinje cells; immature mossy fiber rosettes are present within the internal granule cell layer. A third axon phenotype, beaded ENK fibers can be distinguished on PD 68. Between PD 40 and PD 68, the distributions of ENK climbing and mossy fibers overlap in vermal lobules II–VIII and X, whereas in the hemispheres climbing fibers predominate. However, by PD 83, ENK positive climbing fibers are no longer evident in lateral folia. These results indicate that early arriving ENK axons are present before the differentiation of their cellular targets. Further, a transient appearance of ENK in discrete populations of developing climbing fibers suggests several developmental events: (1) cell death in the inferior olive, (2) collateral regression, or (3) a transient expression of this peptide, that may be characteristic of this chemically defined system of axons.

Heterogeneic expression of blood group A and H isoantigens in bladder tumors: association with nuclear volume.

Intratumor heterogeneity is a major problem in immunodiagnosis and treatment of carcinomas. To elucidate the well-known heterogeneity in transitional-cell carcinomas of the ability to express blood group ABO isoantigens, a stereological estimate of the mean nuclear volume in areas expressing blood group antigens was compared to the estimate from areas of identical pathological grade at which antigen expression was deleted. Four microscopic fields were examined from antigen-positive and four from antigen-negative areas in sections from 21 blood group O and 20 blood group A individuals. The sections were stained before examination by an indirect peroxidase method using monoclonal anti-H and anti-A antibodies. The mean nuclear volume increased, as expected, with increasing pathological grade. In blood group O individuals the mean nuclear volume was 241.5 microns 3 in antigen-positive areas and 338.2 microns 3 in antigen-negative areas (2p less than 0.0005) of identical pathological grade. In group A individuals the mean nuclear volume was 217.1 microns 3 in positive areas and 351.1 microns 3 in corresponding negative areas (2p less than 0.0025). The variation in volume parameter was essentially caused by a true variation between tumors (greater than 82%). The results indicate a complex biological mechanism associated with the cellular ability to express blood group antigens.

Expression of cytokeratin in the epithelium of dentigerous cysts and odontogenic keratocysts: an aid to diagnosis.

Sections of tissue embedded in paraffin wax from 18 selected odontogenic cysts were studied both histologically and immunohistochemically with antibodies to cytokeratins using the indirect peroxidase technique. The cysts were divided on a clinical and histological basis into two equal groups comprising dentigerous cysts and odontogenic keratocysts. It was possible to differentiate the two cyst types in every case by the pattern of staining using the monoclonal antibody LP34 for cytokeratins of intermediate molecular weight. The monoclonal antibody CAM 5.2 for cytokeratins of low molecular weight was not discriminatory. Such a clear distinction may prove useful diagnostically in distinguishing between two cysts of similar appearance but very different behaviour.

Localization of Proopiomelanocortin (POMC) Immunoreactive Neurons in the Forebrain of the Pig13

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.