Schwann Cell Phenotype Changes in Aging Human Dental Pulp.

Schwann cells are glial cells that support axonal development, maintenance, defense, and regeneration in the peripheral nervous system. There is limited knowledge regarding the organization, plasticity, and aging of Schwann cells within the dental pulp in adult permanent teeth. The present study sought to relate changes in the pattern of Schwann cell phenotypes between young and old adult teeth with neuronal, immune, and vascular components of the dental pulp. Schwann cells are shown to form a prominent glial network at the dentin-pulp interface, consisting of nonmyelinating and myelinating phenotypes, forming a multicellular neuroimmune interface in association with nerve fibers and dendritic cells. Schwann cell phenotypes are recognized by the expression of S100, glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), Sox10, GAP43, and p75NTR markers. In young adult teeth, a dense population of nonmyelinating Schwann cells projects processes in close association with sensory nerve terminals through the odontoblast layer, reaching the adjacent predentin/dentin domain. While GAP43 and p75NTR are highly expressed in nonmyelinating Schwann cells from young adult teeth, the presence of these markers declines significantly in old adult teeth. Myelinated axons, identified by MBP expression, are mainly present at the Raschkow plexus and within nerve bundles in the dental pulp, but their density is significantly reduced in old adult versus young adult teeth. These data reveal age-related changes within the glial network of the dental pulp, in association with a reduction of coronal dental pulp innervation in old adult versus young adult teeth. The prominence of Schwann cells as a cellular component at the dentin-pulp interface supports the notion that their association with sensory nerve terminals and immune system components forms part of an integrated multicellular barrier for defense against pathogens and dentin repair.

NO signaling in retinal bipolar cells.

Nitric oxide (NO) is a neuromodulator involved in physiological and pathological processes in the retina. In the inner retina, a subgroup of amacrine cells have been shown to synthesize NO, but bipolar cells remain controversial as NO sources. This study correlates NO synthesis in dark-adapted retinas, through labeling with the NO marker DAF-FM, with neuronal nitric oxide synthase (nNOS) and inducible NOS expression, and presence of the NO receptor soluble guanylate cyclase in bipolar cells. NO containing bipolar cells were morphologically identified by dialysis of DAF fluorescent cells with intracellular dyes, or by DAF labeling followed by immunohistochemistry for nNOS and other cellular markers. DAF fluorescence was observed in all types of bipolar cells that could be identified, but the most intense DAF fluorescence was observed in bipolar cells with severed processes, supporting pathological NO signaling. Among nNOS expressing bipolar cells, type 9 was confirmed unequivocally, while types 2, 3a, 3b, 4, 5, 7, 8 and the rod bipolar cell were devoid of this enzyme. These results establish specific bipolar cell types as NO sources in the inner retina, and support the involvement of NO signaling in physiological and pathological processes in the inner retina.

Axonal Degeneration in Dental Pulp Precedes Human Primary Teeth Exfoliation.

The dental pulp in human primary teeth is densely innervated by a plethora of nerve endings at the coronal pulp-dentin interface. This study analyzed how the physiological root resorption (PRR) process affects dental pulp innervation before exfoliation of primary teeth. Forty-four primary canine teeth, classified into 3 defined PRR stages (early, middle, and advanced) were fixed and demineralized. Longitudinal cryosections of each tooth were stained for immunohistochemical and quantitative analysis of dental pulp nerve fibers and associated components with confocal and electron microscopy. During PRR, axonal degeneration was prominent and progressive in a Wallerian-like scheme, comprising nerve fiber bundles and nerve endings within the coronal and root pulp. Neurofilament fragmentation increased significantly during PRR progression and was accompanied by myelin degradation and a progressive loss of myelinated axons. Myelin sheath degradation involved activation of autophagic activity by Schwann cells to remove myelin debris. These cells expressed a sequence of responses comprising dedifferentiation, proliferative activity, GAP-43 overexpression, and Büngner band formation. During the advanced PRR stage, increased immune cell recruitment within the dental pulp and major histocompatibility complex (MHC) class II upregulation by Schwann cells characterized an inflammatory condition associated with the denervation process in preexfoliative primary teeth. The ensuing loss of dental pulp axons is likely to be responsible for the progressive reduction of sensory function of the dental pulp during preexfoliative stages.

Reactionary Dentinogenesis and Neuroimmune Response in Dental Caries.

Reactionary dentin formation is an adaptive secretory response mediated by odontoblasts to moderate dentin injury. The implications of this process for neuroimmune interactions operating to contain pathogens have not been fully appreciated. The purpose of the present study was to describe the relationship between reactionary dentinogenesis, the neurogenic changes of dental pulp innervation, and dendritic cell recruitment to caries progression, using a comparative immunohistochemical approach in human teeth from young adult individuals. Reactionary dentin formation during dentin caries progression is associated with changes in the integrity of junctional complexes within the odontoblast layer. Diminished coexpression of Cx43 and zonula occludens 1 implies a reduced level of intercellular connectivity between odontoblasts. Dentin caries also causes overexpression of growth-associated protein 43, a modulator of neural plasticity that promotes extensive sprouting of nerve endings into the reactionary dentin matrix. At the same time, an elevated number of HLA-DR-positive dendritic cells infiltrate the odontoblast layer and subsequently invade reactionary dentin formed underneath the early caries-affected regions. Simultaneous odontoblast layer remodeling, nerve fiber sprouting, and activation of dendritic cells during caries progression suggest a coordinated neuroimmune response to fight caries pathogen invasion and to promote dentin-pulp healing. We propose that reactionary dentin formation hinders pathogen invasion and supports defensive neuroimmune interactions against infection. The eventual understanding of this complex scenario may contribute to the development of novel approaches to dental caries treatment.

The amazing odontoblast: activity, autophagy, and aging.

Odontoblasts are dentin-secreting cells that survive for the whole life of a healthy tooth. Once teeth are completely erupted, odontoblasts transform into a mature stage that allows for their functional conservation for decades, while maintaining the capacity for secondary and reactionary dentin secretion. Odontoblasts are also critically involved in the transmission of sensory stimuli from the dentin-pulp complex and in the cellular defense against pathogens. Their longevity is sustained by an elaborate autophagic-lysosomal system that ensures organelle and protein renewal. However, progressive dysfunction of this system, in part caused by lipofuscin accumulation, reduces the fitness of odontoblasts and eventually impairs their dentin maintenance capacity. Here we review the functional activities assumed by mature odontoblasts throughout life. Understanding the biological basis of age-related changes in human odontoblasts is crucial to improving tooth preservation in the elderly.

Calcium-activated chloride channels do not contribute to the odorant transduction current in the marine teleost Isacia conceptionis.

This study compared the contribution of the Ca²âº-activated Cl⁻ conductance to the electroolfactogram (EOG) evoked by different odorant classes between the marine Cabinza grunt Isacia conceptionis and rainbow trout Oncorhynchus mykiss. The Ca²âº-activated Cl⁻ channel blocker niflumic acid significantly diminished odorant responses in O. mykiss, but had no effect on the EOG in I. conceptionis, supporting the notion that Ca²âº-activated Cl⁻ channels may not operate as odorant transduction current amplifiers in this marine teleost.

Mitochondrial autophagy and lipofuscin accumulation in aging odontoblasts.

Aging of long-lived post-mitotic cells is characterized as a progressive and irreversible reduction of functional activity. In such cells, mitochondria are organelles critical for bioenergetic supply, whose turnover is mediated by an autophagic-lysosomal pathway. In human teeth, odontoblasts are post-mitotic cells responsible for sensory function and dentin preservation. Here, human odontoblasts were processed for immunohistochemistry with antibodies against mitochondrial (MTCO2) and lysosomal (LAMP2) markers, and comparatively analyzed in two age groups (young-adult and adult) with light and electron microscopy. Selective engulfment of mitochondrial profiles into autophagic vacuoles is common in young-adult odontoblasts, suggesting a microautophagic pathway. With age, the odontoblast layer is reduced in width, and mitochondrial elements converge around large clusters of autofluorescent lipofuscin deposits. Age-related changes in odontoblasts are observed as a long-term process in which the progressive accumulation of intralysosomal debris limits the autophagic turnover of mitochondrial components, causing an eventual decline in physiological cell functions, which leads to increased vulnerability under stress conditions.

Autophagic activity and aging in human odontoblasts.

Odontoblasts are long-lived post-mitotic cells in the dental pulp, whose function is to form and maintain dentin. The survival mechanisms that preserve the viability of terminally differentiated odontoblasts during the life of a healthy tooth have not been described. In the present study, we characterized the autophagic-lysosomal system of human odontoblasts with transmission electron microscopy and immunocytochemistry, to analyze the mechanisms that maintain the functional viability of these dentinogenic cells. Odontoblasts were found to develop an autophagic-lysosomal system organized mainly by large autophagic vacuoles that are acid-phosphatase-positive to various degrees. These vacuoles expressed the autophagosomal and lysosomal markers LC3 and LAMP2, respectively, in an age-related pattern indicating the organization of a dynamic autophagic machinery. Progressive accumulation of lipofuscin within lysosomes indicates reduced lysosomal activity as a function of odontoblast aging. Our results suggest that autophagic activity in odontoblasts is a fundamental mechanism to ensure turnover and degradation of subcellular components. A reduction in the efficacy of this system might compromise cell viability and dentinogenic secretory capacity. In adult teeth, this condition is described as an 'old odontoblast' stage.

NADPH diaphorase is developmentally regulated in rat olfactory epithelium.

In vertebrate olfactory receptor neurons, NO synthase (NOS) has been detected in embryonic and early postnatal stages. However, expression of the enzyme in the mature epithelium is still controversial. We analyzed the developmental expression pattern of the histochemical NOS-marker NADPH diaphorase (NADPHd) in the olfactory epithelium of young rats. NADPHd was expressed in a small subset of olfactory receptor neurons as early as P0. Between P0 and P24 the number of labeled neurons increased 10-fold, stabilizing thereafter. Whereas NADPHd was generally found in the somata, a transitory dendritic expression was observed between P2 and P5. This dynamic postnatal regulation of the cellular distribution of NADPHd appears to reflect developmental processes within the olfactory epithelium.

Excitation, inhibition, and suppression by odors in isolated toad and rat olfactory receptor neurons.

Vertebrate olfactory receptor neurons (ORNs) exhibit odor-induced increases in action potential firing rate due to an excitatory cAMP-dependent current. Fish and amphibian ORNs also give inhibitory odor responses, manifested as decreases in firing rate, but the underlying mechanism is poorly understood. In the toad, an odor-induced Ca(2+)-activated K(+) current is responsible for the hyperpolarizing receptor potential that causes inhibition. In isolated ORNs, a third manner by which odors affect firing is suppression, a direct and nonspecific reduction of voltage-gated and transduction conductances. Here we show that in whole cell voltage-clamped toad ORNs, excitatory or inhibitory currents were not strictly associated to a particular odorant mixture. Occasionally, both odor effects, in addition to suppression, were concurrently observed in a cell. We report that rat ORNs also exhibit odor-induced inhibitory currents, due to the activation of a K(+) conductance closely resembling that in the toad, suggesting that this conductance is widely distributed among vertebrates. We propose that ORNs operate as complex integrator units in the olfactory epithelium, where the first events in the process of odor discrimination take place.

Calcium mediates the NO-induced potassium current in toad and rat olfactory receptor neurons.

Nitric oxide (NO) activates a K(+) current in dissociated amphibian olfactory receptor neurons. Using the patch-clamp technique in its whole-cell mode and stimulation with puffs of the NO-donor sodium nitroprusside, we further studied this effect and show that it was sensitive to the K(+)-channel blockers tetraethylammonium and iberiotoxin, indicating the activation of a Ca(2+)-dependent K(+) conductance. The Ca(2+)-channel blockers nifedipine and cadmium abolished the NO-induced current, and lowering external Ca(2+) reduced it significantly. Ca(2+) imaging showed a transient fluorescence increase upon stimulation with NO, and after blockade of K(+) currents, an NO-induced inward current could be measured, suggesting that the activation of the Ca(2+)-dependent K(+) conductance is mediated by Ca(2+) influx. LY83583, a blocker of the ciliary cAMP-gated channels, did not affect the current, and experiments with focal stimulation indicated that the effect is present in the soma, therefore Ca(2+) is unlikely to enter via the transduction channels. Finally, we show that NO exerts an effect with similar characteristics on olfactory receptor neurons from the rat. These data represent the first evidence that NO activates a Ca(2+)-dependent K(+) conductance by causing a Ca(2+) influx in a sensory system, and suggest that NO signaling plays a role in the physiology of vertebrate olfactory receptor neurons.

Nitric oxide activates a potassium current in olfactory receptor neurons from Caudiverbera caudiverbera and Xenopus laevis.

The putative role of nitric oxide (NO) in the physiology of olfactory receptor neurons (ORNs) is controversial. Here we report that pulses of NO caused an outward current in voltage-clamped isolated olfactory neurons. The I-V relation of this effect, its sensitivity to charybdotoxin and its dependence on external potassium suggest that NO activates a K(+)-conductance. As blockers of soluble guanylyl cyclases failed to affect the current, we conclude that NO opens K(+)-channels in a cGMP-independent manner.

Nitric oxide and cyclic GMP modulate photoreceptor cell responses in the visual system of the locust

Nitric oxide (NO) is a membrane-permeant messenger molecule which activates the cyclic GMP (cGMP)-synthesizing enzyme soluble guanylyl cyclase. Using cytochemical techniques, we recently reported NO-induced cGMP immunoreactivity in the photoreceptor cells of the compound eye of the locust Schistocerca gregaria and also detected NADPH diaphorase staining, a marker of NO synthase, in a subset of the monopolar cells of the lamina. By recording the corneal electroretinogram (ERG), we found that the application of neurochemicals that raise NO/cGMP levels in the optic lobe increased the ERG amplitude, whereas the experimental reduction of NO levels caused a decrease in the response to light. An increase in the light response was also found in intracellular recordings after application of a NO donor, suggesting that the NO-induced changes in the ERG are not caused by changes in the resistive isolation of the retina. Our cytochemical and electrophysiological data are both consistent with the hypothesis that NO synthesized in monopolar cells is a retrograde messenger to the presynaptic photoreceptor neurones.

Cytochemical evidence for nitric oxide/cyclic GMP signal transmission in the visual system of the locust.

Nitric oxide is a membrane-permeant messenger molecule which activates soluble guanylyl cyclase. Using NADPH diaphorase staining as a marker for the enzyme nitric oxide synthase and an antiserum against cyclic GMP (cGMP) we investigated the possible sites of nitric oxide and cGMP synthesis in the retina and lamina of Schistocerca gregaria. The photoreceptor cells did not express NADPH diaphorase staining but monopolar cells of the lamina were strongly stained. After inhibition of phosphodiesterase activity and incubation of tissue in a nitric oxide donor, the photoreceptor cells showed cGMP immunoreactivity. In contrast to the photoreceptors, the monopolar cells of the lamina were not stained. Since the presynaptic photoreceptors were cGMP-immunoreactive and the postsynaptic targets of the monopolar cells did not express immunoreactivity, it is conceivable that nitric oxide released by monopolar cells may play a role as a retrograde messenger in visual information processing.

The nitric oxide/cyclic GMP messenger system in olfactory pathways of the locust brain.

Nitric oxide is generated by a Ca2+/calmodulin-stimulated nitric oxide synthase and activates soluble guanylyl cyclase. Using NADPH diaphorase (NADPHd) staining as a marker for the enzyme nitric oxide synthase and an antiserum against cGMP, we investigated the cellular organization of nitric oxide donor and target cells in olfactory pathways of the brain of the locust (Schistocerca gregaria). A small subset of neuronal and glial cells expressed cGMP immunoreactivity after incubation of tissue in a nitric oxide donor. Nitric oxide-induced increases in cGMP immunoreactivity were quantified in a tissue preparation of the antennal lobe and in primary mushroom body cell cultures. The mushroom body neuropil is a potential target of a transcellular nitric oxide/cGMP messenger system since it is innervated by extrinsic NADPHd-positive neurons. The mushroom body-intrinsic Kenyon cells do not stain for NADPHd but can be induced to express cGMP immunoreactivity. The colocalization of NADPHd and cGMP immunoreactivity in a cluster of interneurons of the antennal lobe, the principal olfactory neuropil of the insect brain, suggests a role of the nitric oxide/cGMP system in olfactory sensory processing. Colocalization of NADPHd staining and cGMP immunoreactivity was also found in certain glial cells. The cellular organization of the nitric oxide/cGMP system in neurons and glia raises the possibility that nitric oxide acts not only as an intercellular but also as an intracellular messenger molecule in the insect brain.