Hypothalamic detection of macronutrients via multiple gut-brain pathways

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Immunocytochemical demonstration of rod-opsin,S-antigen,and neuron-specific proteins in the human pineal gland

Summary The aim of this study was to examine whether rod-opsin and S-antigen immunoreactions were present in the pineal organ of adult man and how these immunoreactions were correlated with neuronal markers, e.g., synaptophysin, and neurofilaments L, H and M. Three perfusion-fixed epithalamic regions including the pineal organ and five pineal glands obtained at routine autopsy were used. The specimens were taken from female or male patients, 25 to 85 years of age. All immunoreactions were performed using highly specific, well-characterized antibodies. Rod-opsin and S-antigen-immunoreactive pinealocytes occurred in all pineal organs investigated; however, the immunoreaction was restricted to small subpopulations of pinealocytes (rod-opsin immunoreaction: approximately 3%–5%; S-antigen immunoreaction: approximately 5%–10% of the total population). In contrast, immunoreactions for synaptophysin and neurofilaments M and H were present in numerous pinealocytes. Immunoreactivity for neurofilament L was not found. These data suggest that the cellular composition of the human pineal organ is heterogeneous. Moreover, the presence of rod-opsin and S-antigen immunoreactions in the human pineal organ indicates that it may be affected by autoimmune retinal diseases that are provoked by antibodies against these proteins, as is the case in rodents and non-human primates.     

Lipid peroxidation products in human subretinal fluid

The concentrations of thiobarbituric acid reacting substances (TBARS) and proteins in the subretinal fluid (SF) of patients undergoing retinal detachment surgery have been determined. We have tried to establish the correlations between these biochemical and other clinical features of these patients: evolution time of the retinal detachment, age, degree of myopia, and macular affection. Caucasian patients, 19 men and 19 women (57.42 ±12.85 average age, interval 20–80) were randomly selected for this study. SF samples were obtained by puncture after scleral indentation. TBARS and protein concentrations were determined by the corresponding colorimetric assays. A linear correlation exists between TBARS and protein contents in these samples. No correlation could be established between evolution time of the retinal detachment and TBARS content in SF. TBARS in SF increases with increasing age in nonmyopic patients. In the samples of myopic patients the correlation was established between TBARS content and degree of myopia. The group of patients with more than 10 dioptres show a significant higher TBARS concentration in SF than any of the other groups studied. It can be concluded that lipid peroxidation products in SF originate, at least partially, from rod outer segments, and that lipid peroxidation is a process that might play a role in the pathogenesis of retinal detachment, specially in myopic patients.     

Nonlinear spatial integration in retinal bipolar cells shapes the encoding of artificial and natural stimuli

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X-Ray Microanalysis and Phagocytotic Activity of Cultured Retinal Pigment Epithelial Cells in Hypoxia

Purpose: To investigate the influence of the functional and morphological changes induced in retinal pigment epithelial (RPE) cells by retinal ischemia, we evaluated the phagocytotic activity, the concentration of various elements, and ultrastructure in cultured RPE cells in hypoxia. Methods: The concentrations of oxygen in incubators were adjusted to 20, 10, and 1% by the addition of nitrogen for 72 hr. To observe phagocytotic activity and its relationship to actin filaments, the filaments of RPE cells incubated with fluoresbrite carboxylate YG microspheres were stained with rhodamine phalloidin. Some of the specimens were subjected to X-ray microanalysis by scanning electron microscope after being fixed, freeze-dried, and coated with carbon to investigate the cytoplasmic concentration of elements. A part of the latter specimens was also observed by transmission electron microscope after being embedded in epon and cut into ultrathin sections to see the ultra-structural changes inside cell. Results: Lowering oxygen concentrations from 20% to 1% swelled RPE cells and decreased the number of fluoresbrite carboxylate YG microspheres phagocytized by RPE cells. Phagocytosis of a large amount of latex beads (30 μl) for 24 hr in 1% oxygen caused a disruption of RPE cells. Na, S, and P were detected in RPE cells cultured in 20% oxygen. Reducing the oxygen concentration from 20 to 10 or 1% significantly decreased Na and increased S. Mitochondria were observed in RPE cells in 20 and 10% oxygen, but many vacuoles were observed in the cytoplasm in 1% oxygen. Conclusion: Hypoxia as low as 1% oxygen induced malfunction of phagocytosis and the fragility of RPE cells. We could speculate the imbalance of the electrolytes such as Na or a decrease of antioxidants such as glutathione containing S as a reason of disturbance of cell viability.     

Bile acids induce Ca2+ signaling and membrane permeabilizations in vagal nodose ganglion neurons

Bile acids (BAs) play an important role in the digestion of dietary fats and act as signaling molecules. However, due to their solubilizing properties, high concentrations in the gut may negatively affect gut epithelium and possibly afferent fibers innervating the gastrointestinal tract (GI). To determine the effect of BAs on intracellular Ca²⁺ and membrane permeabilization we tested a range of concentrations of two BAs on vagal nodose ganglion (NG) neurons, Chinese Hamster Ovary (CHO), and PC12 cell lines. NG explants from mice were drop-transduced with the genetically encoded Ca²⁺ indicator AAV9-Syn-jGCaMP7s and used to measure Ca²⁺ changes upon application of deoxycholic acid (DCA) and taurocholic acid (TCA). We found that both BAs induced a Ca²⁺ increase in NG neurons in a dose-dependent manner. The DCA-induced Ca²⁺ increase was dependent on intracellular Ca²⁺ stores. NG explants, with an intact peripheral part of the vagus nerve, showed excitation of NG neurons in nerve field recordings upon exposure to DCA. The viability of NG neurons at different BA concentrations was determined, and compared to CHO and PC12 cells lines using propidium iodide labeling, showing threshold concentrations of BA-induced cell death at 400–500 μM. These observations suggest that BAs act as Ca²⁺-inducing signaling molecules in vagal sensory neurons at low concentrations, but induce cell death at higher concentrations, which may occur during inflammatory bowel diseases.     

Hydrogen-bonding network at the cytoplasmic region of a light-driven sodium pump rhodopsin KR2

Light-driven sodium-pumping rhodopsins are able to actively transport sodium ions. Structure/function studies of Krokinobacter eikastus rhodopsin 2 (KR2) identified N61 and G263 at the cytoplasmic surface constituting the “Ion-selectivity filter” for sodium ions, while retinal Schiff base acts as the light “Switch and Gate” for transport of sodium ions. Q123 is located between the two regions, and plays an important role for the pump function, which was implicated by functional, spectroscopic, X-ray crystallographic and computational studies. According to the atomic structure of KR2, Q123 is involved in the hydrogen-bonding network at the cytoplasmic region, together with S64, protein-bound waters, and peptide carbonyl of K255 bound to the chromophore. To gain the detailed structural information around Q123, here we compared light-induced difference Fourier-transform infrared (FTIR) spectra at 77?K between the wild-type (WT) and mutant proteins of KR2, such as Q123A, Q123V, and S64A. The obtained spectra were very similar between WT and these mutants, whereas the observed mutation effects enabled us to identify vibrations of the hydrogen-bonding network at the Q123 and S64 region. This is unique for KR2, not for the corresponding mutations in a light-driven proton-pump bacteriorhodopsin (BR). Hydrogen-bonding alteration is absent for the mutants of KR2, suggesting that proper inter-helical connectivity of helices B, C, and G is important for protein structural changes for sodium-pump function, which is controlled by the region around Q123.     

Retinal damage by light: Possible implication of singlet oxygen

A new hypothesis is proposed in an attempt to explain the mechanism of the irreversible damage which can be induced in the retina by visible light. Upon illumination, retinal generates singlet oxygen and this reactive species can produce lipid peroxidation which in turn may induce membrane instability.Presented at the EMBO-Workshop on Transduction Mechanism of Photoreceptors, Jülich, Germany, October 4–8, 1976