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The retina consumes massive amounts of energy, yet its metabolism and substrate exploitation remain poorly understood. Here, we used a murine explant model to manipulate retinal energy metabolism under entirely controlled conditions and utilised 1H-NMR spectroscopy-based metabolomics, in situ enzyme detection, and cell viability readouts to uncover the pathways of retinal energy production. Our experimental manipulations resulted in varying degrees of photoreceptor degeneration, while the inner retina and retinal pigment epithelium were essentially unaffected. This selective vulnerability of photoreceptors suggested very specific adaptations in their energy metabolism. Rod photoreceptors were found to rely strongly on oxidative phosphorylation, but only mildly on glycolysis. Conversely, cone photoreceptors were dependent on glycolysis but insensitive to electron transport chain decoupling. Importantly, photoreceptors appeared to uncouple glycolytic and Krebs-cycle metabolism via three different pathways: (1) the mini-Krebs-cycle, fuelled by glutamine and branched chain amino acids, generating N-acetylaspartate; (2) the alanine-generating Cahill-cycle; (3) the lactate-releasing Cori-cycle. Moreover, the metabolomics data indicated a shuttling of taurine and hypotaurine between the retinal pigment epithelium and photoreceptors, likely resulting in an additional net transfer of reducing power to photoreceptors. These findings expand our understanding of retinal physiology and pathology and shed new light on neuronal energy homeostasis and the pathogenesis of neurodegenerative diseases.
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Ciclo del Ácido Cítrico , Glucólisis , Fosforilación Oxidativa , Retina , Animales , Ratones , Retina/metabolismo , Metabolismo Energético , Metabolómica , Epitelio Pigmentado de la Retina/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Ratones Endogámicos C57BL , Células Fotorreceptoras Retinianas Conos/metabolismoRESUMEN
Retinal bipolar and amacrine cells receive visual information from photoreceptors and participate in the first steps of image processing in the retina. Several studies have suggested the operation of aerobic glycolysis and a lactate shuttle system in the retina due to the high production of this metabolite under aerobic conditions. However, whether bipolar cells form part of this metabolic circuit remains unclear. Here, we show that the monocarboxylate transporter 2 is expressed and functional in inner retinal neurons. Additionally, we used genetically encoded FRET nanosensors to demonstrate the ability of inner retinal neurons to consume extracellular lactate as an alternative to glucose. In rod bipolar cells, lactate consumption allowed cells to maintain the homeostasis of ions and electrical responses. We also found that lactate synthesis and transporter inhibition caused functional alterations and an increased rate of cell death. Overall, our data shed light on a notable but still poorly understood aspect of retinal metabolism.
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Ácido Láctico , Transportadores de Ácidos Monocarboxílicos , Células Bipolares de la Retina , Animales , Ratones , Metabolismo Energético , Glucosa/metabolismo , Ácido Láctico/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Células Bipolares de la Retina/metabolismoRESUMEN
Purpose: Members of the secretin/glucagon family have diverse roles in retinal physiological and pathological conditions. Out of them, glucagon has been associated with eye growth regulation and image defocus signaling in the eye, both processes central in myopia induction. On the other hand, dopamine is perhaps the most studied molecule in myopia and has been proposed as fundamental in myopia pathogenesis. However, glucagonergic activity in the mammalian retina and its possible link with dopaminergic signaling remain unknown. Methods: To corroborate whether glucagon and dopamine participate together in the modulation of synaptic activity in the retina, inhibitory post-synaptic currents were measured in rod bipolar cells from retinal slices of wild type and negative lens-exposed mice, using whole cell patch-clamp recordings and selective pharmacology. Results: Glucagon produced an increase of inhibitory post-synaptic current frequency in rod bipolar cells, which was also dependent on dopaminergic activity, as it was abolished by dopamine type 1 receptor antagonism and under scotopic conditions. The effect was also abolished after 3-week negative lens-exposure but could be recovered using dopamine type 1 receptor agonism. Conclusions: Altogether, these results support a possible neuromodulatory role of glucagon in the retina of mammals as part of a dopaminergic activity-dependent synaptic pathway that is affected under myopia-inducing conditions.
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Dopamina , Miopía , Animales , Ratones , Dopamina/farmacología , Agonistas de Dopamina/farmacología , Antagonistas de Dopamina , Glucagón , Receptores de Dopamina D1 , Retina , Células Fotorreceptoras Retinianas BastonesRESUMEN
Diabetic retinopathy causes progressive and irreversible damage to the retina through activation of inflammatory processes, overproduction of oxidative species, and glial reactivity, leading to changes in neuronal function and finally ischemia, edema, and hemorrhages. Current treatments are invasive and mostly applied at advanced stages, stressing the need for alternatives. To this end, we tested two unconventional and potentially complementary non-invasive treatment options: Photobiomodulation, the stimulation with near-infrared light, has shown promising results in ameliorating retinal pathologies and insults in several studies but remains controversial. Boldine, on the other hand, is a potent natural antioxidant and potentially useful to prevent free radical-induced oxidative stress. To establish a baseline, we first evaluated the effects of diabetic conditions on the retina with immunofluorescence, histological, and ultrastructural analysis in two diabetes model systems, obese LepRdb/db mice and organotypic retinal explants, and then tested the potential benefits of photobiomodulation and boldine treatment in vitro on retinal explants subjected to high glucose concentrations, mimicking diabetic conditions. Our results suggest that the principal subcellular structures affected by these conditions were mitochondria in the inner segment of photoreceptors, which displayed morphological changes in both model systems. In retinal explants, lactate metabolism, assayed as an indicator of mitochondrial function, was altered, and decreased photoreceptor viability was observed, presumably as a consequence of increased oxidative-nitrosative stress. The latter was reduced by boldine treatment in vitro, while photobiomodulation improved mitochondrial metabolism but was insufficient to prevent retinal structural damage caused by high glucose. These results warrant further research into alternative and complementary treatment options for diabetic retinopathy.
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Diabetes Mellitus , Retinopatía Diabética , Ratones , Animales , Retinopatía Diabética/tratamiento farmacológico , Retinopatía Diabética/metabolismo , Retina/metabolismo , Antioxidantes/farmacología , Estrés Oxidativo , Glucosa/metabolismo , Diabetes Mellitus/metabolismoRESUMEN
Müller cells, the glial cells of the retina, provide metabolic support for photoreceptors and inner retinal neurons, and have been proposed as source of the significant lactate production of this tissue. To better understand the role of lactate in retinal metabolism, we expressed a lactate and a glucose nanosensor in organotypic mouse retinal explants cultured for 14 days, and used FRET imaging in acute vibratome sections of the explants to study metabolite flux in real time. Pharmacological manipulation with specific monocarboxylate transporter (MCT) inhibitors and immunohistochemistry revealed the functional expression of MCT1, MCT2 and MCT4 in Müller cells of retinal explants. The introduction of FRET nanosensors to measure key metabolites at the cellular level may contribute to a better understanding of heretofore poorly understood issues in retinal metabolism.
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Células Ependimogliales , Transferencia Resonante de Energía de Fluorescencia , Ratones , Animales , Células Ependimogliales/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Retina/metabolismo , Ácido Láctico/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismoRESUMEN
INTRODUCTION: Inflammation of the dental pulp due to caries is a highly prevalent pathology which causes intense pain. Here, we sought to correlate the clinical picture with the histopathology of the affected tissue. The interaction between nociceptive neurons and immune cells is fundamental to regulate the inflammatory response, but little is known about the glial network involved in this process, and its impact on caries pathogenesis. METHODS: This study characterized Schwann cells and other neuroimmune components in human dental pulps with reversible and symptomatic irreversible pulpitis (IP). Twenty eight human teeth were extracted for reasons beyond the scope of this study. Twelve were diagnosed as reversible and symptomatic IP respectively, and 4 as controls. The teeth were decalcified, processed for immunolabeling and analyzed with confocal microscopy. RESULTS: Symptomatic IP was characterized by a significantly higher density of neutrophils, and the release of neutrophil extracellular traps. Between IP and healthy controls, there were significant differences in the density of Schwann cells, macrophages, and neutrophils, in addition to morphological alterations. In IP, Schwann cell arborization extended toward the pulpodentinal interface along with more spindle-shaped cell bodies, while some macrophages displayed a distinct fusiform phenotype. CONCLUSIONS: The dental pulp has a complex multicellular organization and its pulpodentinal interface acts as a barrier in which Schwann and immune cells are distributed strategically to stop the progress of pathogens. A synergistic interaction of Schwann cells with immune cells creates a novel perspective to better understand the role of these glial cells and their active participation in pulpal inflammation.
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Caries Dental , Pulpitis , Humanos , Pulpitis/patología , Pulpa Dental/patología , Caries Dental/patología , Inflamación/patología , FenotipoRESUMEN
Kefir beverage is a probiotic food associated with health benefits, containing probiotic microorganisms and biomolecules produced during fermentation. The microbial composition of these beverages varies among countries, geographical regions, and the substrates, therefore, the characterization of kefir beverages is of great relevance in understanding their potential health-promoting and biotechnological applications. Therefore, this study presents the metagenomic and functional characterization of two Chilean kefir beverages, K02 and K03, through shotgun and amplicon-based metagenomic, microbiological, chemical, and biochemical studies. Results show that both beverages' microbiota were mainly formed by Bacteria (>98%), while Eukarya represented less than 2%. Regarding Bacteria, the most abundant genera were Acetobacter (93.43% in K02 and 80.99% in K03) and Lactobacillus (5.72% in K02 and 16.75% in K03), while Kazachstania was the most abundant genus from Eukarya (42.55% and 36.08% in K02 and K03). Metagenomic analyses revealed metabolic pathways for lactose and casein assimilation, biosynthesis of health-promoting biomolecules, and clusters for antibiotic resistance, quorum sensing communication, and biofilm formation. Enzymatic activities, microbial ß-amyloids, and short-chain fatty acids (acetic acid and propionic acid) were also detected in these beverages. Likewise, both kefir beverages inhibited biofilm formation of the opportunistic pathogen Pseudomonas aeruginosa.
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The retina has the highest relative energy consumption of any tissue, depending on a steady supply of glucose from the bloodstream. Glucose uptake is mediated by specific transporters whose regulation and expression are critical for the pathogenesis of many diseases, including diabetes and diabetic retinopathy. Here, we used immunofluorescence to show that glucose transporter-2 (GLUT2) is expressed in horizontal cells of the mouse neuroretina in proximity to inner retinal capillaries. To study the function of GLUT2 in the murine retina, we used organotypic retinal explants, cultivated under entirely controlled, serum-free conditions and exposed them to streptozotocin, a cytotoxic drug transported exclusively by GLUT2. Contrary to our expectations, streptozotocin did not measurably affect horizontal cell viability, while it ablated rod and cone photoreceptors in a concentration-dependent manner. Staining for poly-ADP-ribose (PAR) indicated that the detrimental effect of streptozotocin on photoreceptors may be associated with DNA damage. The negative effect of streptozotocin on the viability of rod photoreceptors was counteracted by co-administration of either the inhibitor of connexin-formed hemi-channels meclofenamic acid or the blocker of clathrin-mediated endocytosis dynasore. Remarkably, cone photoreceptors were not protected from streptozotocin-induced degeneration by neither of the two drugs. Overall, these data suggest the existence of a GLUT2-dependent glucose transport shuttle, from horizontal cells into photoreceptor synapses. Moreover, our study points at different glucose uptake mechanisms in rod and cone photoreceptors.
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Transportador de Glucosa de Tipo 2/metabolismo , Glucosa/metabolismo , Células Fotorreceptoras/metabolismo , Células Horizontales de la Retina/metabolismo , Sinapsis/metabolismo , Animales , Transporte Biológico , Ratones , Retina/metabolismoRESUMEN
Diabetic retinopathy (DR) is one of the main causes of vision loss in the working age population. It is characterized by a progressive deterioration of the retinal microvasculature, caused by long-term metabolic alterations inherent to diabetes, leading to a progressive loss of retinal integrity and function. The mammalian retina presents an orderly layered structure that executes initial but complex visual processing and analysis. Gap junction channels (GJC) forming electrical synapses are present in each retinal layer and contribute to the communication between different cell types. In addition, connexin hemichannels (HCs) have emerged as relevant players that influence diverse physiological and pathological processes in the retina. This article highlights the impact of diabetic conditions on GJC and HCs physiology and their involvement in DR pathogenesis. Microvascular damage and concomitant loss of endothelial cells and pericytes are related to alterations in gap junction intercellular communication (GJIC) and decreased connexin 43 (Cx43) expression. On the other hand, it has been shown that the expression and activity of HCs are upregulated in DR, becoming a key element in the establishment of proinflammatory conditions that emerge during hyperglycemia. Hence, novel connexin HCs blockers or drugs to enhance GJIC are promising tools for the development of pharmacological interventions for diabetic retinopathy, and initial in vitro and in vivo studies have shown favorable results in this regard.
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Conexinas/metabolismo , Retinopatía Diabética/etiología , Retinopatía Diabética/metabolismo , Susceptibilidad a Enfermedades , Uniones Comunicantes/metabolismo , Animales , Conexinas/genética , Retinopatía Diabética/patología , Uniones Comunicantes/genética , Expresión Génica , Humanos , Neuroglía/metabolismo , Retina/metabolismo , Retina/patologíaRESUMEN
Pannexin-1 (Panx1) forms plasma membrane channels that allow the exchange of small molecules between the intracellular and extracellular compartments, and are involved in diverse physiological and pathological responses in the nervous system. However, the signaling mechanisms that induce their opening still remain elusive. Here, we propose a new mechanism for Panx1 channel activation through a functional crosstalk with the highly Ca2+ permeable α7 nicotinic acetylcholine receptor (nAChR). Consistent with this hypothesis, we found that activation of α7 nAChRs induces Panx1-mediated dye uptake and ATP release in the neuroblastoma cell line SH-SY5Y-α7. Using membrane permeant Ca2+ chelators, total internal reflection fluorescence microscopy in SH-SY5Y-α7 cells expressing a membrane-tethered GCAMP3, and Src kinase inhibitors, we further demonstrated that Panx1 channel opening depends on Ca2+ signals localized in submembrane areas, as well as on Src kinases. In turn, Panx1 channels amplify cytosolic Ca2+ signals induced by the activation of α7 nAChRs, by a mechanism that seems to involve ATP release and P2X7 receptor activation, as hydrolysis of extracellular ATP with apyrase or blockage of P2X7 receptors with oxidized ATP significantly reduces the α7 nAChR-Ca2+ signal. The physiological relevance of this crosstalk was also demonstrated in neuroendocrine chromaffin cells, wherein Panx1 channels and P2X7 receptors contribute to the exocytotic release of catecholamines triggered by α7 nAChRs, as measured by amperometry. Together these findings point to a functional coupling between α7 nAChRs, Panx1 channels and P2X7 receptors with physiological relevance in neurosecretion.
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Células Cromafines/metabolismo , Conexinas/metabolismo , Exocitosis/fisiología , Proteínas del Tejido Nervioso/metabolismo , Receptor Cross-Talk/fisiología , Receptores Purinérgicos P2X7/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Animales , Quelantes del Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Bovinos , Línea Celular Tumoral , Células Cromafines/efectos de los fármacos , Exocitosis/efectos de los fármacos , Humanos , Ratones , Receptor Cross-Talk/efectos de los fármacosRESUMEN
Purpose: In the mammalian retina, cannabinoid type 1 receptors (CB1Rs) are well-positioned to alter inhibitory synaptic function from amacrine cells and, thus, might influence visual signal processing in the inner retina. However, it is not known if CB1R modulates amacrine cells feedback inhibition at retinal bipolar cell (BC) terminals. Methods: Using whole-cell voltage-clamp recordings, we examined the pharmacological effect of CB1R activation and inhibition on spontaneous inhibitory postsynaptic currents (sIPSCs) and glutamate-evoked IPSCs (gIPSCs) from identified OFF BCs in light-adapted rat retinal slices. Results: Activation of CB1R with WIN55212-2 selectively increased the frequency of GABAergic, but not glycinergic sIPSC in types 2, 3a, and 3b OFF BCs, and had no effect on inhibitory activity in type 4 OFF BCs. The increase in GABAergic activity was eliminated in axotomized BCs and can be suppressed by blocking CB1R with AM251 or GABAA and GABAρ receptors with SR-95531 and TPMPA, respectively. In all OFF BC types tested, a brief application of glutamate to the outer plexiform layer elicited gIPSCs comprising GABAergic and glycinergic components that were unaffected by CB1R activation. However, blocking CB1R selectively increased GABAergic gIPSCs, supporting a role for endocannabinoid signaling in the regulation of glutamate-evoked GABAergic inhibitory feedback to OFF BCs. Conclusions: CB1R activation shape types 2, 3a, and 3b OFF BC responses by selectively regulate GABAergic feedback inhibition at their axon terminals, thus cannabinoid signaling might play an important role in the fine-tuning of visual signal processing in the mammalian inner retina.
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Receptor Cannabinoide CB1/fisiología , Células Bipolares de la Retina/fisiología , Células Amacrinas/metabolismo , Células Amacrinas/fisiología , Animales , Benzoxazinas/farmacología , Polaridad Celular/efectos de los fármacos , Polaridad Celular/fisiología , Endocannabinoides/metabolismo , Retroalimentación Fisiológica/efectos de los fármacos , Retroalimentación Fisiológica/fisiología , Femenino , Antagonistas de Receptores de GABA-A/farmacología , Ácido Glutámico/farmacología , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/fisiología , Masculino , Morfolinas/farmacología , Naftalenos/farmacología , Técnicas de Placa-Clamp/métodos , Ácidos Fosfínicos/farmacología , Piperidinas/farmacología , Pirazoles/farmacología , Piridinas/farmacología , Ratas Sprague-Dawley , Receptor Cannabinoide CB1/antagonistas & inhibidores , Receptor Cannabinoide CB1/efectos de los fármacos , Retina , Células Bipolares de la Retina/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Spiders are a large group of arthropods and nearly omnipresent in warm and temperate climates. They rely on tactile and visual information to hunt and breed, but compared with their mechanical senses, little is known about their visual systems. In this study, we analyzed the visual spectral sensitivity of the Chilean recluse spider Loxosceles laeta, a synanthropic species posing a significant threat to humans, using electroretinogram recordings of its three eye types and open field tests with localized chromatic illumination for behavioral analysis. The electroretinogram displayed two sensitivity peaks in the ultraviolet and green ranges, and no differences were observed between the three eye types and between male and female specimens. Selective chromatic adaptation reduced overall light sensitivity, but did not support the expression of more than one type of rhodopsin in photoreceptors. The open field tests revealed a preference for corners over side areas, and an increased exploration of open field areas illuminated by shorter wavelength (violet to green) light compared with non-illuminated areas, while no behavioral responses to red and near-infrared light were observed. These data suggest that L. laeta has monochromatic vision without spectral specializations in its three secondary eye pairs.
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Percepción de Color , Arañas/fisiología , Animales , Femenino , MasculinoRESUMEN
One of the tissues of the central nervous system most affected by diabetes is the retina. Despite a growing understanding of the biochemical processes involved in glucose toxicity, little is known about the physiological consequences of chronic high glucose (HG) on individual neurons and neuronal circuits. Electroretinogram recordings suggest that retinal bipolar cells (BCs), which filter and transmit photoreceptor output to the inner retina, are among the first cells affected by diabetic conditions, and may therefore serve as sensitive early biomarkers for incipient neuronal damage caused in diabetes. Here, we comparatively assessed retinal integrity, calcium responses, and the electrophysiological profiles of specific BC types of mouse and rat organotypic retinal explants after 1 to 3 weeks in tissue culture, under moderate glucose (MG) and HG conditions. While the retinal layers of both rodent species displayed a progressively reduced thickness in culture, BCs retained their electrophysiological profiles and remained morphologically identifiable for up to 2 weeks. Responses to glutamate and endogenous inhibitory responses were routinely observed, indicating that the retinal circuitry remained intact during this period. Significant physiological differences between MG and HG conditions were evident in calcium signals and in the time course of responses to glutamate, but the voltage-gated current profiles of BCs displayed only minor variations. Overall, rat retina appeared slightly more sensitive to HG levels compared with mouse. In conclusion, electrophysiological analysis of neuronal function in rodent retinal explants is useful for the study of early damage due to HG neurotoxicity.
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Glucosa/toxicidad , Síndromes de Neurotoxicidad/fisiopatología , Retina/efectos de los fármacos , Retina/fisiopatología , Animales , Retinopatía Diabética/fisiopatología , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Sprague-DawleyRESUMEN
About half of all extant vertebrates are teleost fishes. Although our knowledge about anatomy and function of their olfactory systems still lags behind that of mammals, recent advances in cellular and molecular biology have provided us with a wealth of novel information about the sense of smell in this important animal group. Its paired olfactory organs contain up to five types of olfactory receptor neurons expressing OR, TAAR, VR1- and VR2-class odorant receptors associated with individual transduction machineries. The different types of receptor neurons are preferentially tuned towards particular classes of odorants, that are associated with specific behaviors, such as feeding, mating or migration. We discuss the connections of the receptor neurons in the olfactory bulb, the differences in bulbar circuitry compared to mammals, and the characteristics of second order projections to telencephalic olfactory areas, considering the everted ontogeny of the teleost telencephalon. The review concludes with a brief overview of current theories about odor coding and the prominent neural oscillations observed in the teleost olfactory system.
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Mammalian teeth have evolved as dentin units that enclose a complex system of sensory innervation to protect and preserve their structure and function. In human dental pulp (DP), mechanosensory and nociceptive fibers form a dense meshwork of nerve endings at the coronal dentin-pulp interface, which arise from myelinated and non-myelinated axons of the Raschkow plexus (RP). Schwann cells (SCs) play a crucial role in the support, maintenance and regeneration after injury of these fibers. We have recently characterized two SC phenotypes hierarchically organized within the coronal and radicular DP in human teeth. Myelinating and non-myelinating SCs (nmSCs) display a high degree of plasticity associated with nociceptive C-fiber sprouting and axonal degeneration in response to DP injuries from dentin caries or physiological root resorption (PRR). By comparative immunolabeling, confocal and electron microscopy, we have characterized short-term adaptive responses of SC phenotypes to nerve injuries, and long-term changes related to aging. An increase of SCs characterizes the early responses to caries progression in association with axonal sprouting in affected DP domains. Moreover, during PRR, the formation of bands of Büngner is observed as part of SC repair tracks functions. On the other hand, myelinated axon density is significantly reduced with tooth age, as part of a gradual decrease in DP defense and repair capacities. The remarkable plasticity and capacity of SCs to preserve DP innervation in different dental scenarios constitutes a fundamental aspect to improve clinical treatments. This review article discusses the central role of myelinating and non-mSCs in long-term tooth preservation and homeostasis.
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A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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A17 amacrine cells are an important part of the scotopic pathway. Their synaptic varicosities receive glutamatergic inputs from rod bipolar cells (RBC) and release GABA onto the same RBC terminal, forming a reciprocal feedback that shapes RBC depolarization. Here, using patch-clamp recordings, we characterized electrical coupling between A17 cells of the rat retina and report the presence of strongly interconnected and non-coupled A17 cells. In coupled A17 cells, evoked currents preferentially flow out of the cell through GJs and cross-synchronization of presynaptic signals in a pair of A17 cells is correlated to their coupling degree. Moreover, we demonstrate that stimulation of one A17 cell can induce electrical and calcium transients in neighboring A17 cells, thus confirming a functional flow of information through electrical synapses in the A17 coupled network. Finally, blocking GJs caused a strong decrease in the amplitude of the inhibitory feedback onto RBCs. We therefore propose that electrical coupling between A17 cells enhances feedback onto RBCs by synchronizing and facilitating GABA release from inhibitory varicosities surrounding each RBC axon terminal. GJs between A17 cells are therefore critical in shaping the visual flow through the scotopic pathway.
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Células Amacrinas/fisiología , Células Bipolares de la Retina/metabolismo , Animales , Calcio/metabolismo , Adaptación a la Oscuridad/fisiología , Retroalimentación , Femenino , Uniones Comunicantes/fisiología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Visión Nocturna/fisiología , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Receptores AMPA/metabolismo , Receptores de GABA/metabolismo , Retina/metabolismo , Retina/fisiología , Células Bipolares de la Retina/fisiología , Células Fotorreceptoras Retinianas Bastones/fisiología , Sinapsis/metabolismo , Transmisión Sináptica/fisiología , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Spiders of the family Sicariidae pose a serious threat to affected populations, and Loxosceles laeta (Nicolet) is considered the most venomous species. Development of nontoxic olfaction-based spider repellents or traps is hindered by a current lack of knowledge regarding olfactory system function in arachnids. In the present study, general plant odorants and conspecific odors were tested for behavioral responses in L. laeta. Although general odorants triggered neither attraction nor aversion, conspecific odor of the opposite sex caused aversion in females, and attraction in males. These results support the presence of a specific olfactory system for the detection of conspecifics in L. laeta, but suggest the absence of a broadly tuned system for general odorant detection in this species.
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Quimiotaxis , Odorantes , Percepción Olfatoria , Arañas/fisiología , Animales , Chile , Femenino , MasculinoRESUMEN
Although connexins (Cxs) are broadly expressed by cells of mammalian organisms, Cx39 has a very restricted pattern of expression and the biophysical properties of Cx39-based channels [hemichannels (HCs) and gap junction channels (GJCs)] remain largely unknown. Here, we used HeLa cells transfected with Cx39 (HeLa-Cx39 cells) in which intercellular electrical coupling was not detected, indicating the absence of GJCs. However, functional HCs were found on the surface of cells exposed to conditions known to increase the open probability of other Cx HCs (e.g., extracellular divalent cationic-free solution (DCFS), extracellular alkaline pH, mechanical stimulus and depolarization to positive membrane potentials). Cx39 HCs were blocked by some traditional Cx HC blockers, but not by others or a pannexin1 channel blocker. HeLa-Cx39 cells showed similar resting membrane potentials (RMPs) to those of parental cells, and exposure to DCFS reduced RMPs in Cx39 transfectants, but not in parental cells. Under these conditions, unitary events of ~75 pS were frequent in HeLa-Cx39 cells and absent in parental cells. Real-time cellular uptake experiments of dyes with different physicochemical features, as well as the application of a machine-learning approach revealed that Cx39 HCs are preferentially permeable to molecules characterized by six categories of descriptors, namely: (1) electronegativity, (2) ionization potential, (3) polarizability, (4) size and geometry, (5) topological flexibility and (6) valence. However, Cx39 HCs opened by mechanical stimulation or alkaline pH were impermeable to Ca2+. Molecular modeling of Cx39-based channels suggest that a constriction present at the intracellular portion of the para helix region co-localizes with an electronegative patch, imposing an energetic and steric barrier, which in the case of GJCs may hinder channel function. Results reported here demonstrate that Cx39 form HCs and add to our understanding of the functional roles of Cx39 HCs under physiological and pathological conditions in cells that express them.
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Retinal bipolar cells (BCs) divide photoreceptor output into different channels for the parallel extraction of temporal and chromatic stimulus properties. In rodents, five types of OFF BCs have been differentiated, based on morphological and functional criteria, but their electrophysiological characterization remains incomplete. This study analyzed OFF BCs with the patch clamp technique in acute slices of rat retina. Their specific voltage-dependent currents and glutamate responses are shown to represent individual fingerprints which define the signal processing and filtering properties of each cell type and allow their unequivocal identification. Two additions to the rat BC repertoire are presented: OFF BC-2', a variation of BC-2 with wider axonal arbours and prominent Na(+) currents, is described for the first time in rodents, and OFF BC-3b, previously identified in mouse, is electrophysiologically characterized in rat. Moreover, the glutamate responses of rat OFF BCs are shown to be differentially sensitive to AMPA- and kainate-receptor blockers and to modulation by nitric oxide (NO) through a cGMP-dependent mechanism. These results contribute to our understanding of the diversity and function of bipolar cells in mammals.