E. Viña

Acid-sensing ion channels in healthy and degenerated human intervertebral disc

Abstract Acid-sensing ion channels (ASICs) are a family of H+-gated voltage-insensitive ion channels that respond to extracellular acidification by regulating transmembrane Ca2+ flux. Moreover, ASICs can also be gated by mechanical forces and may function as mechanosensors. The cells of the intervertebral disc (IVD) have an unusual acidic and hyperosmotic microenvironment. Changes in the pH and osmolarity determine the viability of IVD cells and the composition of the extracellular matrix, and both are the basis of IVD degeneration. In this study, the expression of ASICs (ASIC1, ASIC2, ASIC3 and ASIC4) mRNAs and proteins in human healthy and degenerated IVD was evaluated by quantitative reverse transcription-quantitative polymerase chain reaction and Western blot. The distribution of ASIC proteins was determined by immunohistochemistry. The mRNAs for all ASICs were detected in normal human IVD, and significantly increased levels were found in degenerated IVD. Western blots demonstrated the presence of proteins with estimated molecular weights of approximately 68–72 kDa. In both the annulus fibrosus (AF) and nucleus pulposus (NP) of normal IVD, ASIC2 is the most frequently expressed ASIC followed by ASIC3, ASIC1 and ASIC4. In the AF of degenerated IVD, there was a significant increase in the number of ASIC1 and ASIC4 positive cells, whereas in the NP, we found significant increase of expression of ASIC1, ASIC2 and ASIC3. These results describe the occurrence and localization of different ASICs in human healthy IVD, and their increased expression in degenerated IVD, thus suggesting that ASICs may be involved in IVD degeneration.

Acid-sensing ion channels (ASICs) in the taste buds of adult zebrafish.

In detecting chemical properties of food, different molecules and ion channels are involved including members of the acid-sensing ion channels (ASICs) family. Consistently ASICs are present in sensory cells of taste buds of mammals. In the present study the presence of ASICs (ASIC1, ASIC2, ASIC3 and ASIC4) was investigated in the taste buds of adult zebrafish (zASICs) using Western blot and immunohistochemistry. zASIC1 and zASIC3 were regularly absent from taste buds, whereas faint zASIC2 and robust zASIC4 immunoreactivities were detected in sensory cells. Moreover, zASIC2 also immunolabelled nerves supplying taste buds. The present results demonstrate for the first time the presence of zASICs in taste buds of teleosts, with different patterns to that occurring in mammals, probably due to the function of taste buds in aquatic environment and feeding. Nevertheless, the role of zASICs in taste remains to be demonstrated.

Acid-sensing ion channel 2 (ASIC2) is selectively localized in the cilia of the non-sensory olfactory epithelium of adult zebrafish.

Abstract Ionic channels play key roles in the sensory cells, such as transducing specific stimuli into electrical signals. The acid-sensing ion channel (ASIC) family is voltage-insensitive, amiloride-sensitive, proton-gated cation channels involved in several sensory functions. ASIC2, in particular, has a dual function as mechano- and chemo-sensor. In this study, we explored the possible role of zebrafish ASIC2 in olfaction. RT-PCR, Western blot, chromogenic in situ hybridization and immunohistochemistry, as well as ultrastructural analysis, were performed on the olfactory rosette of adult zebrafish. ASIC2 mRNA and protein were detected in homogenates of olfactory rosettes. Specific ASIC2 hybridization was observed in the luminal pole of the non-sensory epithelium, especially in the cilia basal bodies, and immunoreactivity for ASIC2 was restricted to the cilia of the non-sensory cells where it was co-localized with the cilia marker tubulin. ASIC2 expression was always absent in the olfactory cells. These findings demonstrate for the first time the expression of ASIC2 in the olfactory epithelium of adult zebrafish and suggest that it is not involved in olfaction. Since the cilium sense and transduce mechanical and chemical stimuli, ASIC2 expression in this location might be related to detection of aquatic environment pH variations or to detection of water movement through the nasal cavity.

Calcium-activated potassium channel SK1 is widely expressed in the peripheral nervous system and sensory organs of adult zebrafish

Sensory cells contain ion channels involved in the organ-specific transduction mechanisms that convert different types of stimuli into electric energy. Here we focus on small-conductance calcium-activated potassium channel 1 (SK1) which plays an important role in all excitable cells acting as feedback regulators in after-hyperpolarization. This study was undertaken to analyze the pattern of expression of SK1 in the zebrafish peripheral nervous system and sensory organs using RT-PRC, Westernblot and immunohistochemistry. Expression of SK1 mRNA was observed at all developmental stages analyzed (from 10 to 100 days post fertilization, dpf), and the antibody used identified a protein with a molecular weight of 70kDa, at 100dpf (regarded to be adult). Cell expressing SK1 in adult animals were neurons of dorsal root and cranial nerve sensory ganglia, sympathetic neurons, sensory cells in neuromasts of the lateral line system and taste buds, crypt olfactory neurons and photoreceptors. Present results report for the first time the expression and the distribution of SK1 in the peripheral nervous system and sensory organs of adult zebrafish, and may contribute to set zebrafish as an interesting experimental model for calcium-activated potassium channels research. Moreover these findings are of potential interest because the potential role of SK as targets for the treatment of neurological diseases and sensory disorders.

Acid-sensing ion channels (ASICs) 2 and 4.2 are expressed in the retina of the adult zebrafish

Acid-sensing ion channels (ASICs) are H+-gated, voltage-insensitive cation channels involved in synaptic transmission, mechanosensation and nociception. Different ASICs have been detected in the retina of mammals but it is not known whether they are expressed in adult zebrafish, a commonly used animal model to study the retina in both normal and pathological conditions. We study the expression and distribution of ASIC2 and ASIC4 in the retina of adult zebrafish and its regulation by light using PCR, in situ hybridization, western blot and immunohistochemistry. We detected mRNA encoding zASIC2 and zASIC4.2 but not zASIC4.1. ASIC2, at the mRNA or protein level, was detected in the outer nuclear layer, the outer plexiform layer, the inner plexiform layer, the retinal ganglion cell layer and the optic nerve. ASIC4 was expressed in the photoreceptors layer and to a lesser extent in the retinal ganglion cell layer. Furthermore, the expression of both ASIC2 and ASIC4.2 was down-regulated by light and darkness. These results are the first demonstration that ASIC2 and ASIC4 are expressed in the adult zebrafish retina and suggest that zebrafish could be used as a model organism for studying retinal pathologies involving ASICs.

Immunohistochemical characterization of the crypt neurons in the olfactory epithelium of adult zebrafish.

The fish sensory epithelium contains three types of sensory cells denominated ciliated, microvillous, and crypt neurons. Each one differs from the other in its morphological, ultrastructural and molecular features, as well as in their projections to the central nervous system. Crypt neurons are present in both bony and cartilaginous fish and can be identified on the basis of their morphology and the expression of some specific proteins and genes. In this study we have investigated the morphology of crypt neurons, as well as the occurrence and co-localization of S100 protein, calretinin and TRPV4, three proposed markers for crypt cells, in the olfactory epithelium of adult zebrafish (Danio rerio) using double immunofluorescence associated to laser confocal microscopy. A sparse population of superficial S100 protein positive cells was detected being identified as crypt neurons. The calretinin immunoreactive cells were more abundant, occasionally resembling the morphology of the crypt cells but never displaying co-localization of both proteins. The TRPV4 positive cells differed in morphology from crypt cells, thus excluding the occurrence of TRPV4 in those cells. These results demonstrate that only S100 protein immunoreactivity can be used to identify crypt cells. Because some calretinin positive cells showed localization and morphology similar to the crypt cells of the sensory epithelium, the occurrence of two subtypes of crypt cells, S100 protein and calretinin positive, cannot be excluded. The significance of these findings remains to be elucidated.

Acid-sensing ion channels and transient-receptor potential ion channels in zebrafish taste buds

Sensory information from the environment is required for life and survival, and it is detected by specialized cells which together make up the sensory system. The fish sensory system includes specialized organs that are able to detect mechanical and chemical stimuli. In particular, taste buds are small organs located on the tongue in terrestrial vertebrates that function in the perception of taste. In fish, taste buds occur on the lips, the flanks, and the caudal (tail) fins of some species and on the barbels of others. In fish taste receptor cells, different classes of ion channels have been detected which, like in mammals, presumably participate in the detection and/or transduction of chemical gustatory signals. However, since some of these ion channels are involved in the detection of additional sensory modalities, it can be hypothesized that taste cells sense stimuli other than those specific for taste. This mini-review summarizes current knowledge on the presence of transient-receptor potential (TRP) and acid-sensing (ASIC) ion channels in the taste buds of teleosts, especially adult zebrafish. Up to now ASIC4, TRPC2, TRPA1, TRPV1 and TRPV4 ion channels have been found in the sensory cells, while ASIC2 was detected in the nerves supplying the taste buds.

Chondroitin Sulfate in Human Cutaneous Meissner and Pacinian Sensory Corpuscles: Extracellular matrix in cutaneous corpuscles

Chondroitin sulfate is a glycosaminoglycan involved in maintaining the morphofunctional properties of the extracellular matrix in peripheral nerves, but its distribution in human sensory corpuscles is unknown despite the role of extracellular matrix in mechanotransduction and axonal guidance. In this study we used immunohistochemistry to analyze the distribution of chondroitin sulfate in human cutaneous Meissner and Pacinian corpuscles. Chondroitin sulfate expression was absent from Meissner corpuscles. In Pacinian corpuscles chondroitin sulfate was found associated to a CD34 positive endoneurial‐related layer, interposed between the S100 protein positive inner core cells, and the vimentin positive inner core and outer core‐capsule cells. Therefore, the intermediate CD34+/chondroitin sulfate+ intermediate layer present in Pacinian corpuscles isolates the neural segment of the corpuscles (axon and inner core) from the non‐neural segments (outer core and capsule). These results suggest a role of chondroitin sulfate in the proper axonal growth and guidance, within the neuronal compartment of the Pacinian corpuscles during development and reinnervation, can be hypothesized. Moreover, a role of CS in mechanotransduction cannot be ruled out. Anat Rec, 302:325–331, 2019.