Morphologic characterization of rat taste receptor cells that express components of the phospholipase C signaling pathway

Rat taste buds contain three morphologically distinct cell types that are candidates for taste transduction. The physiologic roles of these cells are, however, not clear. Inositol 1,4,5-triphosphate (IP(3)) has been implicated as an important second messenger in bitter, sweet, and umami taste transductions. Previously, we identified the type III IP(3) receptor (IP(3)R3) as the dominant isoform in taste receptor cells. In addition, a recent study showed that phospholipase Cbeta(2) (PLCbeta(2)) is essential for the transduction of bitter, sweet, and umami stimuli. IP(3)R3 and PLCbeta(2) are expressed in the same subset of cells. To identify the taste cell types that express proteins involved in PLC signal transduction, we used 3,3'diaminobenzidine tetrahydrochloride immunoelectron microscopy and fluorescence microscopy to identify cells with IP(3)R3. Confocal microscopy was used to compare IP(3)R3 or PLCbeta(2) immunoreactivity with that of some known cell type markers such as serotonin, protein gene-regulated product 9.5, and neural cell adhesion molecule. Here we show that a large subset of type II cells and a small subset of type III cells display IP(3)R3 immunoreactivity within their cytoplasm. These data suggest that type II cells are the principal transducers of bitter, sweet, and umami taste transduction. However, we did not observe synapses between type II taste cells and nerve fibers. Interestingly, we observed subsurface cisternae of smooth endoplasmic reticulum at the close appositions between the plasma membrane of type II taste cells and nerve processes. We speculate that some type II cells may communicate to the nervous system via subsurface cisternae of smooth endoplasmic reticulum in lieu of conventional synapses.     

Catecholamine-independent transient expression of tyrosine hydroxylase in primary auditory neurons is coincident with the onset of hearing in the rat cochlea

During the last stages of neuronal maturation, tyrosine hydroxylase is transiently expressed in the absence of the other catecholamine-synthesizing enzymes. We show here that it is expressed in rat spiral ganglion neurons between postnatal days 8 and 20, with a peak of expression at postnatal day 12. These tyrosine hydroxylase-immunoreactive neurons did not display aromatic amino acid decarboxylase- or dopamine-beta-hydroxylase-immunoreactivities, ruling out the possibilities of dopamine or noradrenaline synthesis. They also did not display peripherin- or intense neurofilament 200-kDa-immunoreactivities, two indicators of type II primary auditory neurons. Tyrosine hydroxylase-immunoreactive dendrites were seen in synaptic contact with the inner hair cells and expressed the GluR2 subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors, further confirming the type I nature of the neurons transiently expressing the enzyme. The end of the tyrosine hydroxylase expression was not due to cell death because the immunoreactive neurons did not show TUNEL-labelled nuclei. Finally, all the type I neurons expressed the tyrosine hydroxylase mRNA at postnatal day 12, suggesting that the expression of the enzyme is a maturational step common to all these neurons and that the expression of the protein is not synchronized. Because the period of transient expression of tyrosine hydroxylase in type I neurons parallels the periods of maturation of evoked exocytosis in inner hair cells and of appearance and maturation of the cochlear potentials, we propose that the expression of the enzyme indicates the onset of hearing in individual type I primary auditory neurons. This enzyme expression could rely on a Ca2+ activation of its encoding gene subsequent to a sudden and massive Ca2+ entry through voltage-activated Ca2+ channels.     

Double Labeling Immulloelectron Microscopic Study on the Synaptic Connections between Glutamic Acid Neurons and GABA Neurons in the Hippocampus of Rats

The pathogenesis of epilepsy is a very complicated process, In which neurotransmltters play important roles. The hippocamplls is a frequently attacked site and an ideal model tissue for epilepsy' l]. In general, the seizure results from the imbalance of excitation and inhibition, i. e., increased effect of excltatory neurotransmltters ordecreased effect of inhlbitory neurotransrnlttersL =' ']. However, there was no nlorphologlcal, eel)eclally at the ultrastructural level, evidenceic identify …     

电惊厥大鼠海马生长抑素神经元的超微结构变化

观察电惊厥大鼠海马生长抑素神经元的超微结构变化，为探讨生长抑素在癫痫发病中的作用机制提供形态学依据。采用电惊厥癫痫动物模型和免疫电镜方法观察。结果：电惊厥时海马内生长抑素神经元胞体和突起出现不同程度的结构损伤，表现为线粒体肿胀、细胞器崩解和神经末梢变性等；在海马区多形细胞层和分子层生长抑素神经元胞体、突起与非生长抑素神经元胞体、突起之间有复杂的突触联系。结论：电惊厥时海马CA4生长抑素神经元结构发生损伤。     

Attractin/mahogany protein expression in the rodent central nervous system

Attractin/Mahogany protein (Atrn) is known to be involved in a number of physiological and neuropathological events. Although the ubiquitous distribution of atrn mRNA has been described in neurons, lack of detailed information concerning the cellular and subcellular localization of protein product is impeding understanding of the role of Atrn. The present study immunohistochemically examined distributions of Atrn in rat and mouse central nervous systems (CNSs) by using a novel antibody for Atrn. Atrn was intensely expressed in most neurons and dendrites of large neurons, such as cortical pyramidal neurons and cerebellar Purkinje neurons. Intense Atrn expression was also observed in the neuropil of gray matter in many regions of the CNS, such as the main and accessory olfactory bulb, cerebral cortex, caudate putamen, dorsal lateral geniculate nucleus, medial eminence, superior colliculus, hippocampus, dentate gyrus, and layers 1 and 2 of the spinal cord. Furthermore, we found that astrocytes, microglia, and ependymal cells also express Atrn protein. Immunoelectron microscopy showed the subcellular distribution of Atrn in the plasma membrane of cell soma, dendrites, and spines in neurons and in the cytoplasmic membrane of Golgi apparatus, endoplasmic reticulum, and mitochondria in neurons and glial cells. These findings indicate that Atrn is more widely expressed throughout the CNS than previously reported, and expression of Atrn by various cell types suggests that Atrn may serve multiple functions in the CNS.     

Variable expression of tenascin‐C,osteopontin and fibronectin in inflammatory myofibroblastic tumour of the lung

Kaarteenaho R, Sormunen R, Pääkkö P. Variable expression of tenascin‐C, osteopontin and fibronectin in inflammatory myofibroblastic tumour of the lung. APMIS 2010; 118: 91–100. The aim of this study was to analyse the expression of tenascin‐C, osteopontin and fibronectin in inflammatory myofibroblastic tumour of the lung, which is a rare tumour of unknown aetiology. Nine patients with an inflammatory myofibroblastic tumour of lung were studied by immunohistochemistry for the presence of tenascin‐C, osteopontin, fibronectin and alpha‐smooth muscle actin, which is a common marker for myofibroblasts. The ultrastructure of myofibroblasts was confirmed by transmission electron microscopy. The expression of tenascin‐C, osteopontin, fibronectin and alpha‐smooth muscle actin was also studied by immunoelectron microscopy. All cases displayed all of the studied extracellular matrix proteins and also alpha‐smooth muscle actin‐positive spindle‐shaped fibroblastic cells that were undoubtedly myofibroblasts. The immunoelectron microscopic studies demonstrated labelling for alpha‐smooth muscle actin in intracellular filament bundles within myofibroblasts, for fibronectin in the extracellular filaments of the fibronexus and for tenascin‐C extracellularly often adjacent to myofibroblasts. Labels for osteopontin were observed within myofibroblasts and plasma cells. These results demonstrate that tenascin‐C, osteopontin and fibronectin were expressed in all three kinds of subtypes of inflammatory myofibroblastic tumours of the lung and further, variable amounts of myofibroblasts could be observed by light and transmission electron microscopy as well as by immunoelectron microscopic techniques.     

Adenocarcinoma of Rectosigmoid Colon

A case of hybrid leukemia is presented. A 30-year-old man had two blast cell populations with bone marrow. The majority of the blast cells had B-lymphoid markers on their surfaces and a small number of cells had both lymphoid markers and penoxidase activity on the same cell. Immunoelectron microscopy combined with ultrastructural cytochemistry (MPO reaction) demonstrated the biphenotypic nature of the blast cells.     

Single Stress Induces Long-Lasting Elevations in Vasopressin mRNA Levels in CRF Hypophysiotrophic Neurones, but Repeated Stress is Required to Modify AVP Immunoreactivity

Repeated stress is known to induce an increased vasopressin (AVP) expression in paraventricular corticotrophin-releasing factor (CRF) neurones which is supposed to enhance the ACTH-releasing capacity of these cells. To test the hypothesis that a single stress is sufficient to produce these changes, we used quantitative in-situ hybridization analysis to measure steady state CRF and AVP mRNA. Moreover the colocalized AVP and CRF immunoreactive sites were assessed in the dense core vesicle compartment of CRF axon terminals in the external zone of the median eminence with quantitative immunoelectron microscopy. Acute immobilization produced a significant increase in the average AVP and CRF mRNA levels (145% and 65%, respectively, above control values) in the medial parvocellular subdivisions of the paraventricular nucleus (PVN), and these changes persisted for over 4 days after stress. In contrast to these changes in AVP mRNA levels, there were no concomitant changes in AVP immunostaining in CRF terminals and axons during the 4-day period. However, when immobilization stress was repeated daily, the number of CRF terminals containing AVP increased progressively. Moreover, the ratio of AVP and CRF immunoreactivity in the dense core vesicle compartment was increased. Taken together, these results provide evidence that single stress experience can cause long-lasting changes in AVP and CRF mRNA steady state expression that is not apparently accompanied by changes in peptide levels. They also suggest that repeated stress is required for developing progressive shifts in the neurohormone storage pattern of these neurones.     

Comparative immunoelectron microscopy with monoclonal antibodies on yellow fever virus-infected cells: pre-embedding labelling versus immunocryoultramicrotomy

To study the intra- and extracellular distribution of yellow fever virus 17D (YFV)-specific antigens, pre-embedding immunoelectron microscopy (IEM) and IEM on ultrathin frozen sections were carried out comparatively using monoclonal antibodies (MAB) and YFV-infected cells. In addition, three electron-dense marker systems (IgG-ferritin and IgG-gold and protein A-gold) were compared for their efficiency in detecting bound MAB. Pre-embedding immuno-labelling was performed in microtest plates followed by in situ embedding and immunocryoultramicrotomy was performed using pellets of sucrose-infused cells. In both procedures, cells were prefixed with different concentrations of glutaraldehyde (GA). In pre-embedding IEM virus-specific antigens could be detected on the envelopes of extracellular virions with YFV-neutralizing MAB. Using immunocryoultramicrotomy, neutralizing MAB bound to intracellular mature virions as well as to viral antigens incorporated into cytoplasmic membranes. A concentration of 1% GA destroyed antigenicity entirely, while with 0.25% and 0.1% GA immunoreactivity was retained for more than 3 mth. Some highly reactive MAB labelled antigen significantly in pre-embedding IEM, when used at concentrations of 1 ng/ml. Immunocryoultramicrotomy was 10-100 times less sensitive. On cryosections colloidal gold was the marker of choice, due to the fact that it showed less nonspecific sticking to intracellular components and that it was easily detectable on highly contrasted cryosections. Owing to their higher sensitivity, IgG-ferritin conjugates were preferred in pre-embedding IEM.