Immunocytochemical Techniques Research Articles

A small round cell lesion in the neck of an 11-year-old child

Small round cell lesions in the neck of a pediatric patient may involve a lymph node or present as an extra-nodal solid tumor. Various proliferative lymph node lesions include non-specific reactive hyperplasia, infectious mononucleosis, Castleman’s disease, Rosai-Dorfman disease, Langerhans cell histiocytosis and malignant lymphoma. Besides lymphomas, the other pediatric small round cell tumors (SRCT) of the neck can be a group of malignancies that include neuroblastoma, Ewing’s sarcoma, embryonal rhabdomyosarcoma, and metastasis from retinoblastoma and small cell undifferentiated carcinoma. These lesions are characterized both histologically and cytologically by predominantly small round to oval cells. Special morphological features characterizing these lesions are exclusive noncohesive cells and lymphoglandular bodies for non-Hodgkin lymphoma; rosette formation and filamentous background for neuroblastoma; nuclear molding, acinar formation and paranuclear blue inclusion for metastatic small cell undifferentiated carcinoma; glycogen vacuoles for Ewing’s sarcoma; and tadpole cells for embryonal rhabdomyosarcoma. However, the so called characteristic cytomorphologic features may not be obvious or may be observed in more than one SRCT. To solve the differential diagnostic problems, ancillary studies like cytochemistry, immunocytochemistry, ultrastructure, flow cytometry, cytogenetics and molecular techniques may be of help. The present case is that of a small round cell lesion of neck in an 11-year-old girl.

Neuropeptides in the antennal lobe of the yellow fever mosquito, Aedes aegypti

For many insects, including mosquitoes, olfaction is the dominant modality regulating their behavioral repertoire. Many neurochemicals modulate olfactory information in the central nervous system, including the primary olfactory center of insects, the antennal lobe. The most diverse and versatile neurochemicals in the insect nervous system are found in the neuropeptides. In the present study, we analyzed neuropeptides in the antennal lobe of the yellow fever mosquito, Aedes aegypti, a major vector of arboviral diseases. Direct tissue profiling of the antennal lobe by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry indicated the presence of 28 mature products from 10 different neuropeptide genes. In addition, immunocytochemical techniques were used to describe the cellular location of the products of up to seven of these genes within the antennal lobe. Allatostatin A, allatotropin, SIFamide, FMRFamide-related peptides, short neuropeptide F, myoinhibitory peptide, and tachykinin-related peptides were found to be expressed in local interneurons and extrinsic neurons of the antennal lobe. Building on these results, we discuss the possible role of neuropeptide signaling in the antennal lobe of Ae. aegypti. J. Comp. Neurol. 522:592–608, 2014.

Oligoprogenitor Cells Derived from Spermatogonia Stem Cells Improve Remyelination in Demyelination Model

Embryonic stem (ES) like cells-derived testis represents a possible alternative to replace of neurons and glia. Here, we differentiated spermatogonia cells to oligoprogenitor (OP) like cells and transplanted them to demyelination model and assess their recovery potential in a demyelinated corpus callosum model in rats. ES like cells were differentiated to OP like cells using appropriate inducers and were transplanted in situ to demyelinated corpus callosum. Cell integration as well as demyelination extension and myelination intensity changes were evaluated using histologic studies and immunocytochemistry after 2 and 4 weeks post transplantation. Investigation of Nestin, NF68, Olig2, and NG2 by immunocytochemical technique indicated the differentiation of ES like cells to neuroprogenitor and oligodendrocyte like cells in each induction stage. Histologic findings showed a significant decrease in demyelination extension and a significant increase in remyelination intensity in cell transplanted groups. Also on the base of PLP expression, differentiation of transplanted cells was confirmed to myelinogenic cells using immunocytochemistry technique. We conclude that ES like cells derived from spermatogonia cells can be differentiated to OP like cells that can form myelin after transplantation into the demyelination model in rat, this represents recovery potential of spermatogonia cells which opens new window for cell therapeutic approaches using spermatogonial stem cells.

Biophysical characteristics of cells cultured on cholesteryl ester liquid crystals

This study aimed at examining the biophysical characteristics of human derived keratinocytes (HaCaT) cultured on cholesteryl ester liquid crystals (CELC). CELC was previously shown to improve sensitivity in sensing cell contractions. Characteristics of the cell integrin expressions and presence of extracellular matrix (ECM) proteins on the liquid crystals were interrogated using various immunocytochemical techniques. The investigation was followed by characterization of the chemical properties of the liquid crystals (LC) after immersion in cell culture media using Fourier transform infrared spectroscopy (FTIR). The surface morphology of cells adhered to the LC was studied using atomic force microscopy (AFM). Consistent with the expressions of the integrins α2, α3 and β1, extracellular matrix proteins (laminin, collagen type IV and fibronectin) were found secreted by the HaCaT onto CELC and these proteins were also secreted by cells cultured on the glass substrates. FTIR analysis of the LC revealed the existence of spectrum assigned to cholesterol and ester moieties that are essential compounds for the metabolizing activities of keratinocytes. The immunostainings indicated that cell adhesion on the LC is mediated by self-secreted ECM proteins. As revealed by the AFM imaging, the constraint in cell membrane spread on the LC leads to the increase in cell surface roughness and thickness of cell membrane. The biophysical expressions of cells on biocompatible CELC suggested that CELC could be a new class of biological relevant material.

PLEURAL MESOTHELIOMA: ETIOLOGY, INCIDENCE, DIAGNOSIS, TREATMENT AND SURVIVAL

The incidence rate of malignant pleural mesothelioma accounts for 0.2 cases per 100,000 population among men and 0.1 cases among women in Russia, and 0.88 and 0.67 cases respectively in Altai Territory. Recently, there has been a growing number of the incidences due to a widespread use of asbestos in the 70th years of the last century. Malignant mesothelioma is distinguished by a prolonged latency period, and constitutes a highly aggressive tumor. By now, the diagnosis of pleural mesothelioma still remains a problem. CT and US-scans are used together with chest X-ray. Morphological confirmation of tumor (biopsy and cytologic examination of exudation complemented by immunocytochemical technique) is obligatory. Treatment of malignant mesothelioma mostly includes chemotherapy with cisplatin and pemetrexed. Radical surgery is more frequently impossible considering a diffusion growth of mesothelioma. Radiotherapy is used as prevention, and applied to a puncture point and post-surgery to prevent local recurrence. The life expectancy of the patients with malignant pleural is poor: from 4 to 12 months with an average of 7 months. Only 26.5% of such patients have life expectancy longer than one year, and 6.8% of such patients live longer than 3 years. Maximum life expectancy of patients with malignant pleural mesothelioma accounts for 44 months. These are women with accumulation of fluid diagnosed in the left-sided pleural space with epithelioid mesothelioma.

Towards the characterization of an in vitro triple co-culture intestine cell model for permeability studies

Caco-2 based cell models have been the gold standard in vitro method to study intestinal drug permeability, despite the absence of many important features with major influence in the drug absorption mechanism. In the present work, a triple co-culture comprising Caco-2, HT29-MTX and Raji B cells was established to mimic in a closely way the human intestinal epithelium, presenting the main components in the process of drug absorption, namely the absorptive cells that resemble enterocytes, mucus producers cells and cells able to induce M-cell phenotype on Caco-2 cells. All the three cell lines maintained their function when cultured together with each other being, thus, an asset to new orally administrated drugs development. The seeding ratio of 90:10 between Caco-2 and HT29-MTX showed to be the best to achieve physiological proportions after cells maturation and differentiation in culture. The formation of M-cells phenotype from enterocytes was identified for the first time in a co-culture system comprising Caco-2 and HT29-MTX cells through immunocytochemical techniques. Thus, the triple co-culture model presented in the herein work is a good and reliable alternative to the in vitro methods already existents for the study of drugs permeability.

GABAB receptors play an essential role in maintaining sleep during the second half of the night in Drosophila melanogaster

GABAergic signalling is important for normal sleep in humans and flies. Here we advance the current understanding of GABAergic modulation of daily sleep patterns by focusing on the role of slow metabotropic GABAB receptors in the fruit fly Drosophila melanogaster. We asked whether GABAB-R2 receptors are regulatory elements in sleep regulation in addition to the already identified fast ionotropic Rdl GABAA receptors. By immunocytochemical and reporter-based techniques we show that the pigment dispersing factor (PDF)-positive ventrolateral clock neurons (LNv) express GABAB-R2 receptors. Downregulation of GABAB-R2 receptors in the large PDF neurons (l-LNv) by RNAi reduced sleep maintenance in the second half of the night, whereas sleep latency at the beginning of the night that was previously shown to depend on ionotropic Rdl GABAA receptors remained unaltered. Our results confirm the role of the l-LNv neurons as an important part of the sleep circuit in D. melanogaster and also identify the GABAB-R2 receptors as the thus far missing component in GABA-signalling that is essential for sleep maintenance. Despite the significant effects on sleep, we did not observe any changes in circadian behaviour in flies with downregulated GABAB-R2 receptors, indicating that the regulation of sleep maintenance via l-LNv neurons is independent of their function in the circadian clock circuit.

Selenium Compounds Prevent Amyloid β-Peptide Neurotoxicity in Rat Primary Hippocampal Neurons

Neuropathological hallmarks of Alzheimer's disease (AD) include amyloid plaque formation, neurofibrillary tangles, neuronal and synaptic loss. This study aims to identify the neuroprotective effects of the selenium compounds on the neurotoxicity of amyloid β(1-42) in primary cultures of murine hippocampal neurons. Samples were subjected to immunocytochemistry and western blotting techniques to determine the role of treatments on neuronal viability and synaptic protein SNAP-25. We observed a reduced cell viability amyloid β-peptide (1-42)-induced. When cells were co-treated with amyloid β-peptide (1-42) and selenium compounds, we verified a strong increase in relative cell viability and in the level of synaptic marker synaptosomal-associated protein SNAP-25 induced by selenium compounds.

Rapid Immunocytochemistry with Simple Heat-Induced Antigen Retrieval Technique for Improvement in the Quality of Cytological Diagnosis

Rapid immunocytochemistry (ICC) can improve the accuracy of intraoperative cytological diagnoses; however, it is usually applied without heat-induced antigen retrieval (HIAR). We established a HIAR method for rapid ICC and evaluated its efficacy and reliability. Rapidly fixed smear samples were immunostained using 35 antibodies. We compared the results of HIAR by boiling in a pot or heating in an electric kettle. The smears were incubated for 3 min with each primary antibody and immuno-enzyme polymer reagent, and for 1 min with diaminobenzidine solution. HIAR for 1 min using the kettle method yielded the best cellular integrity. For 32 out of the 35 antibodies, results achieved using rapid ICC within 11 min were comparable to that achieved using standard ICC. HIAR was essential for 13 antibodies. For two of the antibodies, HIAR was not required when standard ICC was applied, but consistent staining with rapid ICC was obtained only with HIAR. In conclusion, we established a rapid ICC procedure using a simple HIAR method, which allowed efficient immunostaining of a panel of antigens, including nuclear antigens, within only 11 min. The combined use of this rapid ICC technique with other staining techniques could be useful for improving intraoperative cytological diagnoses.

Validation of cell‐based fluorescence assays: Practice guidelines from the ICSH and ICCS – part I – rationale and aims

Flow cytometry and other technologies of cell‐based fluorescence assays are as a matter of good laboratory practice required to validate all assays, which when in clinical practice may pass through regulatory review processes using criteria often defined with a soluble analyte in plasma or serum samples in mind. Recently the U.S. Food and Drug Administration (FDA) has entered into a public dialogue in the U.S. regarding their regulatory interest in laboratory developed tests (LDTs) or so‐called “home brew” assays performed in clinical laboratories. The absence of well‐defined guidelines for validation of cell‐based assays using fluorescence detection has thus become a subject of concern for the International Council for Standardization of Haematology (ICSH) and International Clinical Cytometry Society (ICCS). Accordingly, a group of over 40 international experts in the areas of test development, test validation, and clinical practice of a variety of assay types using flow cytometry and/or morphologic image analysis were invited to develop a set of practical guidelines useful to in vitro diagnostic (IVD) innovators, clinical laboratories, regulatory scientists, and laboratory inspectors. The focus of the group was restricted to fluorescence reporter reagents, although some common principles are shared by immunohistochemistry or immunocytochemistry techniques and noted where appropriate. The work product of this two year effort is the content of this special issue of this journal, which is published as 5 separate articles, this being Validation of Cell‐based Fluorescence Assays: Practice Guidelines from the ICSH and ICCS ‐ Part I ‐ Rationale and aims. © 2013 International Clinical Cytometry Society

Validation of cell‐based fluorescence assays: practice guidelines from the ICSH and ICCS – part II – preanalytical issues

Flow cytometry and other technologies of cell-based fluorescence assays are as a matter of good laboratory practice required to validate all assays, which when in clinical practice may pass through regulatory review processes using criteria often defined with a soluble analyte in plasma or serum samples in mind. Recently the U.S. Food and Drug Administration (FDA) has entered into a public dialogue in the U.S. regarding their regulatory interest in laboratory developed tests (LDTs) or so-called "home brew" assays performed in clinical laboratories. The absence of well-defined guidelines for validation of cell-based assays using fluorescence detection has thus become a subject of concern for the International Council for Standardization of Haematology (ICSH) and International Clinical Cytometry Society (ICCS). Accordingly, a group of over 40 international experts in the areas of test development, test validation, and clinical practice of a variety of assay types using flow cytometry and/or morphologic image analysis were invited to develop a set of practical guidelines useful to in vitro diagnostic (IVD) innovators, clinical laboratories, regulatory scientists, and laboratory inspectors. The focus of the group was restricted to fluorescence reporter reagents, although some common principles are shared by immunohistochemistry or immunocytochemistry techniques and noted where appropriate. The work product of this two year effort is the content of this special issue of this journal, which is published as 5 separate articles, this being Validation of Cell-based Fluorescence Assays: Practice Guidelines from the ICSH and ICCS - Part II - Preanalytical issues. © 2013 International Clinical Cytometry Society.

Validation of cell‐based fluorescence assays: Practice guidelines from the ICSH and ICCS – part IV – postanalytic considerations

Flow cytometry and other technologies of cell-based fluorescence assays are as a matter of good laboratory practice required to validate all assays, which when in clinical practice may pass through regulatory review processes using criteria often defined with a soluble analyte in plasma or serum samples in mind. Recently the U.S. Food and Drug Administration (FDA) has entered into a public dialogue in the U.S. regarding their regulatory interest in laboratory developed tests (LDTs) or so-called home brew assays performed in clinical laboratories. The absence of well-defined guidelines for validation of cell-based assays using fluorescence detection has thus become a subject of concern for the International Council for Standardization of Haematology (ICSH) and International Clinical Cytometry Society (ICCS). Accordingly, a group of over 40 international experts in the areas of test development, test validation, and clinical practice of a variety of assay types using flow cytometry and/or morphologic image analysis were invited to develop a set of practical guidelines useful to in vitro diagnostic (IVD) innovators, clinical laboratories, regulatory scientists, and laboratory inspectors. The focus of the group was restricted to fluorescence reporter reagents, although some common principles are shared by immunohistochemistry or immunocytochemistry techniques and noted where appropriate. The work product of this two year effort is the content of this special issue of this journal, which is published as 5 separate articles, this being Validation of Cell-based Fluorescence Assays: Practice Guidelines from the ICSH and ICCS - Part IV - Postanalytic considerations.

A novel, highly sensitive method for assessing gap junctional coupling

To assess gap junctional intercellular communication we have developed a tracer-based methodology which is both highly sensitive and potentially adaptable for in vivo measurements. We found that injection of serotonin revealed significantly more intercellular communication than that injection of the most permeant synthetic tracer currently in use, neurobiotin. Furthermore, mechanical tracer loading steps can be replaced by transfection with human serotonin transporter and the inclusion of serotonin in the medium. Tracer and transporter are detected using immunocytochemical techniques and the presence of cells that are tracer-positive but transporter-negative indicates junctional communication. Tracer loading in vivo using transgenesis, electroporation or viral transduction to direct expression of transporter should be more easily accomplished than with mechanical loading methods.

Hamster Oviductin Regulates Tyrosine Phosphorylation of Sperm Proteins During In Vitro Capacitation1

Oviductin or OVGP1, also known as oviduct-specific glycoprotein, has been shown to enhance sperm capacitation in addition to its other beneficial effects on fertilization and early embryo development. We hypothesized that estrus stage-specific hamster oviductin (eHamOVGP1) can potentiate the enhancement of tyrosine phosphorylation of sperm proteins during capacitation. Immunofluorescent staining and confocal microscopy as well as immunocytochemistry and surface replica technique localized tyrosine-phosphorylated proteins to the equatorial segment and midpiece after incubation of hamster sperm in capacitation medium in the presence or absence of eHamOVGP1. Increase of tyrosine phosphorylation level in the equatorial segment occurred as early as 5 min after incubation in the presence of eHamOVGP1. Immunostaining for eHamOVGP1 further increased upon prolonged incubation of sperm in medium containing the glycoprotein. Regardless of the presence or absence of eHamOVGP1, phosphotyrosine expression was observed along the tail, particularly at the midpiece. Western blotting of NP40-extracted sperm proteins (25, 37, and 44 kDa) and NP40-non-extractable sperm proteins (70, 83, 90 kDa) showed increased immunolabeling intensity after 5, 60, 120, and 180 min of capacitation in the presence of eHamOVGP1. Mass spectrometric analysis identified several proteins of functions known to be involved in metabolic pathways responsible for enhancement of tyrosine phosphorylation in its presence. The present investigation provides evidence that eHamOVGP1 regulates the expression of protein tyrosine phosphorylation in sperm capacitated in vitro, further supporting an important role of the presence of OVGP1 in the oviductal milieu during the process of fertilization.

한국관박쥐 망막 원뿔세포의 조직화학적 분석

목적: 한국관박쥐의 망막에서 원뿔세포의 middle/long(ML) opsin cone photoreceptors의 분포를 분석하여 박쥐의 시각계를 이해하고자 하였다. 방법: 표준면역세포화학법을 이용하여 성체 한국관박쥐의 망막을 대상으로 조사하였다. 결과: 4 개체의 망막 전체에서 추정된 ML opsin은 <TEX>$27,336{\pm}2,130$</TEX>개였으며, 평균밀도는 <TEX>$7,854{\pm}268cells/mm^2$</TEX>이었다. S opsin은 외핵층에 위치한 세포외절에서 일부 면역반응성을 보였다. 결론: ML opsin의 조직화된 분포와 S opsin의 발현 결과는 한국관박쥐가 밝은 빛에도 반응하며 색을 구별할 수 있는 기능을 가지고 있다는 것을 알 수 있다. Purpose: This study was done to understand the visual system of bat by analyzing the distribution of middle/long (ML) opsin cone photoreceptors in the retina of the greater horseshoe bat. Methods: Experiments have been performed by standard immunocytochemical techniques on retina of the greater horseshoe bat Rhinolophus ferrumequinum. Results: The estimated numbers of ML cones were <TEX>$27,336{\pm}2,130$</TEX> cells and the mean density of them was <TEX>$7,854{\pm}268cells/mm^2$</TEX> among the four retinas. S opsin was appeared a little immunoreactivity in the outer segments of outer nuclear layer of cones. Conclusions: From the well organized spatial distributions of ML opsin and the immunoreactivity of S opsin in the retinas, the greater horseshoe bats have the functions not only reacting in the photopic vision but being able to distinguish the colors.

Oatp58Dc contributes to blood-brain barrier function by excluding organic anions from the Drosophila brain

The blood-brain barrier (BBB) physiologically isolates the brain from the blood and, thus, plays a vital role in brain homeostasis. Ion transporters play a critical role in this process by effectively regulating access of chemicals to the brain. Organic anion-transporting polypeptides (Oatps) transport a wide range of amphipathic substrates and are involved in efflux of chemicals across the vertebrate BBB. The anatomic complexity of the vascularized vertebrate BBB, however, creates challenges for experimental analysis of these processes. The less complex structure of the Drosophila BBB facilitates measurement of solute transport. Here we investigate a physiological function for Oatp58Dc in transporting small organic anions across the BBB. We used genetic manipulation, immunocytochemistry, and molecular techniques to supplement a whole animal approach to study the BBB. For this whole animal approach, the traceable small organic anion fluorescein was injected into the hemolymph. This research shows that Oatp58Dc is involved in maintaining a chemical barrier against fluorescein permeation into the brain. Oatp58Dc expression was found in the perineurial and subperineurial glia, as well as in postmitotic neurons. We specifically targeted knockdown of Oatp58Dc expression in the perineurial and subperineurial glia to reveal that Oatp58Dc expression in the perineurial glia is necessary to maintain the barrier against fluorescein influx into the brain. Our results show that Oatp58Dc contributes to maintenance of a functional barrier against fluorescein influx past the BBB into the brain.

Regulated forms of cell death are induced by the photodynamic action of the fluorogenic substrate, Hypocrellin B-acetate

The addition of chemical groups to a photosensitizer makes it to act as a fluorogenic substrate, increasing its ability to enter the cells. In this work, the cytotoxic efficacy of Hypocrellin B modified by addition of two acetate groups (HypB-Ac) was investigated in HeLa cells. Using transmission electron microscopy, cytochemical and immunocytochemical techniques, and flow cytometry we demonstrated that light irradiation of HypB-Ac-loaded cells resulted in either necrosis or apoptosis, depending on the HypB-Ac concentration. Administration of Hyp-Ac at high concentration (1×10−5 M) resulted in massive necrosis, while at low concentration (2.5×10−7 M) apoptosis along with autophagy were induced. Focusing on cells still exhibiting non-apoptotic features, we provide the evidence of early involvement of different organelles in the photodamage, with the frequent presence of autophagic vacuoles already at very short post-irradiation times (30 min, when ultrastructural apoptotic features are rarely found). These findings suggest that the widespread photodamage rather than the target organelle(s) involved is crucial for inducing either a catastrophic or a regulated form of cell death. Fluorogenic substrates such as HypB-Ac have an increased capability to accumulate in cancer cells compared to the native photosensitizing molecules: this would allow to use lower drug doses in vivo, thus decreasing the risk of systemic cytotoxicity in the absence of irradiation improving the efficacy of photodynamic therapy. The ability of HypB-Ac at very low concentration to induce autophagy and apoptosis would additionally be advantageous for therapeutic application, as the preferential induction of regulated forms of cell death entails the rapid phagocytotic removal of dying cells without affecting the tissue and organ structure.

Electrophoretic profiling and immunocytochemical detection of pectins and arabinogalactan proteins in olive pollen during germination and pollen tube growth

Cell wall pectins and arabinogalactan proteins (AGPs) are important for pollen tube growth. The aim of this work was to study the temporal and spatial dynamics of these compounds in olive pollen during germination. Immunoblot profiling analyses combined with confocal and transmission electron microscopy immunocytochemical detection techniques were carried out using four anti-pectin (JIM7, JIM5, LM5 and LM6) and two anti-AGP (JIM13 and JIM14) monoclonal antibodies. Pectin and AGP levels increased during olive pollen in vitro germination. (1 → 4)-β-d-Galactans localized in the cytoplasm of the vegetative cell, the pollen wall and the apertural intine. After the pollen tube emerged, galactans localized in the pollen tube wall, particularly at the tip, and formed a collar-like structure around the germinative aperture. (1 → 5)-α-l-Arabinans were mainly present in the pollen tube cell wall, forming characteristic ring-shaped deposits at regular intervals in the sub-apical zone. As expected, the pollen tube wall was rich in highly esterified pectic compounds at the apex, while the cell wall mainly contained de-esterified pectins in the shank. The wall of the generative cell was specifically labelled with arabinans, highly methyl-esterified homogalacturonans and JIM13 epitopes. In addition, the extracellular material that coated the outer exine layer was rich in arabinans, de-esterified pectins and JIM13 epitopes. Pectins and AGPs are newly synthesized in the pollen tube during pollen germination. The synthesis and secretion of these compounds are temporally and spatially regulated. Galactans might provide mechanical stability to the pollen tube, reinforcing those regions that are particularly sensitive to tension stress (the pollen tube-pollen grain joint site) and mechanical damage (the tip). Arabinans and AGPs might be important in recognition and adhesion phenomena of the pollen tube and the stylar transmitting cells, as well as the egg and sperm cells.

LA CYTOPONCTION THYROIDIENNE A L'AIGUILLE FINE : TECHNIQUE, MORPHOLOGIE ET INTERET

This series retrospectively reviewed 357 cases who received thyroid fine needle examination at the pathologi laboratory (C.H.U Ibn Sina) during 11 years (1989-1999). The autors explain technical condition of this fine needle aspiration; elements of microscopical analyses which establish the diagnostic without special immunocytochemistry techniques; and the results.

Hypothalamic Hypocretinergic/Orexinergic Neurons Projecting to the Oral Pontine Rapid Eye Movement Sleep Inducing Site in the Cat

The cat ventral oral pontine reticular nucleus (vRPO) is responsible for the generation and maintenance of rapid eye movement (REM) sleep. Hypothalamic neurons containing the peptide hypocretin-1 (also called orexin-A) which will be herewith defined as orexinergic (Orx) neurons, occupy a pre-eminent place in the integration and stabilization of arousal networks as well as in the physiopathology of narcolepsy/cataplexy. In the previous investigations, low-volume and dose microinjections of hypocretin-1 in cat vRPO produced a specific and significant suppression of REM sleep. The aim of this study is to map the hypothalamic Orx neurons that project to the vRPO and suppress REM sleep generation in the cat. Five adult cats received microinjections of the retrograde tracer cholera toxin (CTb) into the vRPO. Brains were processed employing both CTb staining and antiorexin-A immunocytochemistry techniques. A large number of double-labeled neurons (Orx-CTb) intermingled with the single CTb-positive and single Orx neurons were detected in the ipsilateral lateral, perifornical, dorsal, anterior, perimammillothalamic, and posterior hypothalamic areas but were very scarce in the paraventricular, dorsomedial, ventromedial, and periventricular hypothalamic nuclei. A considerable number of double-labeled neurons were also observed in both the dorsal and the lateral hypothalamic areas in the contralateral hypothalamus. Our results suggest that the widely distributed Orx neuronal hypothalamic groups could physiologically inhibit REM sleep generation in vRPO.