J. Cobo

Characterization of sensory deficits in TrkB knockout mice

The sensory deficit in TrkB deficient mice was evaluated by counting the neuronal loss in lumbar dorsal root ganglia (DRG), the absence of sensory receptors (cutaneous--associated to the hairy and glabrous skin - muscular and articular), and the percentage and size of the neurocalcin-positive DRG neurons (a calcium-binding protein which labels proprioceptive and mechanoceptive neurons). Mice lacking TrkB lost 32% of neurons, corresponding to the intermediate-sized and neurocalcin-positive ones. This neuronal lost was accomplished by the absence of Meissner corpuscles, and reduction of hair follicle-associated sensory nerve endings and Merkel cells. The mutation was without effect on Pacinian corpuscles, Golgis organs and muscle spindles. Present results further characterize the sensory deficit of the TrkB-/- mice demonstrating that the intermediate-sized neurons in lumbar DRG, as well as the cutaneous rapidly and slowly adapting sensory receptors connected to them, are under the control of TrkB for survival and differentiation. This study might serve as a baseline for future studies in experimentally induced neuropathies affecting TrkB positive DRG neurons and their peripheral targets, and to use TrkB ligands in the treatment of neuropathies in which cutaneous mechanoreceptors are primarily involved.

Neurotrophin receptors in taste buds of adult zebrafish (Danio rerio)

TrkB plays crucial roles in the development and maintenance of taste buds in mammals. In this study we investigated the presence and cell localization of Trks (TrkA, TrkB and TrkC) in taste buds of the zebrafish (Danio rerio) using Western blot and immunohistochemistry. Proteins of 140 and 145 kDa, identified as full-length TrkA and TrkB, were detected. Conversely, the anti-TrkC antibody recognized a protein lower than expected (100 kDa). In agreement with these results the sensory cells of taste buds, displayed TrkA- and TrkB-like, but not TrkC-like, immunoreactivity. TrkA and TrkB co-existed in the same taste buds, but remains to be clarified whether or not they are co-expressed in the same cells. Present results demonstrate that as for mammals neurotrophins might play a role in sensory cells of the teleostean taste buds.

Mechanosensory neurons, cutaneous mechanoreceptors, and putative mechanoproteins

The mammalian skin has developed sensory structures (mechanoreceptors) that are responsible for different modalities of mechanosensitivity like touch, vibration, and pressure sensation. These specialized sensory organs are anatomically and functionally connected to a special subset of sensory neurons called mechanosensory neurons, which electrophysiologically correspond with Aβ fibers. Although mechanosensory neurons and cutaneous mechanoreceptors are rather well known, the biology of the sense of touch still remains poorly understood. Basically, the process of mechanosensitivity requires the conversion of a mechanical stimulus into an electrical signal through the activation of ion channels that gate in response to mechanical stimuli. These ion channels belong primarily to the family of the degenerin/epithelium sodium channels, especially the subfamily acid‐sensing ion channels, and to the family of transient receptor potential channels. This review compiles the current knowledge on the occurrence of putative mechanoproteins in mechanosensory neurons and mechanoreceptors, as well as the involvement of these proteins on the biology of touch. Furthermore, we include a section about what the knock‐out mice for mechanoproteins are teaching us. Finally, the possibilities for mechanotransduction in mechanoreceptors, and the common involvement of the ion channels, extracellular membrane, and cytoskeleton, are revisited. Microsc. Res. Tech. 75:1033–1043, 2012.

Differential Localization of Acid-Sensing Ion Channels 1 and 2 in Human Cutaneus Pacinian Corpuscles

Acid-sensing ion channels (ASICs) are the members of the degenerin/epithelial sodium channel (Deg/ENaC) superfamily which mediate different sensory modalities including mechanosensation. ASICs have been detected in mechanosensory neurons as well as in peripheral mechanoreceptors. We now investigated the distribution of ASIC1, ASIC2, and ASIC3 proteins in human cutaneous Pacinian corpuscles using immunohistochemistry and laser confocal-scanner microscopy. We detected different patterns of expression of these proteins within Pacinian corpuscles. ASIC1 was detected in the central axon co-expressed with RT-97 protein, ASIC2 was expressed by the lamellar cells of the inner core co-localized with S100 protein, and ASIC3 was absent. These results demonstrate for the first time the differential distribution of ASIC1 and ASIC2 in human rapidly adapting low-threshold mechanoreceptors, and suggest specific roles of both proteins in mechanotransduction.

TrkB is necessary for the normal development of the lung

Normal development of the lung requires coordinated activation of cascades of signaling pathways initiated by growth factors signaling through their receptors. TrkB and its ligands, brain-derived neurotrophic factor (BDNF) and neurotrophin-4, belong to the neurotrophin family of growth factors, which are expressed in a large variety of non-neuronal tissues including the lung. Aberrant neurotrophin signaling underlies the pathogenesis of several lung-related pathologies, including asthma and lung cancer, however, little is known about the role of neurotrophins in the embryonic development of the lung. To fill this gap in knowledge, we analyzed the pattern of TrkB expression in the murine lung and we observed that TrkB is expressed in alveolar macrophages, type II pneumocytes, neuroepithelial bodies and nerves. Analysis of the structure of lung from mice deficient in TrkB revealed that absence of TrkB signaling results in thinner bronchial epithelium and apparent larger air space, and, more importantly, lack of neuroepithelial bodies, an important reduction in the density of nerve fibres in the bronchial smooth muscle, submucous plexus in bronchioles, and pulmonary artery walls. These findings suggest TrkB is essential for the normal development of the lung and the nervous system in the lung.

TRPV4 in the sensory organs of adult zebrafish

TRPV4 is a nonselective cation channel that belongs to the vanilloid (V) subfamily of transient receptor potential (TRP) ion channels. While TRP channels have been found to be involved in sensing temperature, light, pressure, and chemical stimuli, TPRV4 is believed to be primarily a mechanosensor although it can also respond to warm temperatures, acidic pH, and several chemical compounds. In zebrafish, the expression of trpv4 has been studied during embryonic development, whereas its pattern of TPRV4 expression during the adult life has not been thoroughly analyzed. In this study, the occurrence of TRPV4 was addressed in the zebrafish sensory organs at the mRNA (RT‐PCR) and protein (Westernblot) levels. Once the occurrence of TRPV4 was demonstrated, the TRPV4 positive cells were identified by using immunohistochemistry. TPRV4 was detected in mantle and sensory cells of neuromasts, in a subpopulation of hair sensory cells in the macula and in the cristae ampullaris of the inner ear, in sensory cells in the taste buds, in crypt neurons and ciliated sensory neurons of the olfactory epithelium, and in cells of the retina. These results demonstrate the presence of TRPV4 in all sensory organs of adult zebrafish and are consistent with the multiple physiological functions suspected for TRPV4 in mammals (mechanosensation, hearing, and temperature sensing), but furthermore suggest potential roles in olfaction and vision in zebrafish. Microsc. Res. Tech., 2012.

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 68–72u2009kDa. 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.

Enteric glial cells express full-length TrkB and depend on TrkB expression for normal development.

The embryonic development of the enteric nervous system (ENS) from neural crest precursor cells requires neurotrophic signaling. Neurotrophins (NTs) are a family of growth factors that bind Trk receptors to signal diverse functions, including development and maintenance of different cell populations in the peripheral nervous system. In this study we investigated the expression and cell localization of TrkB, the high affinity receptor for brain-derived neurotrophic factor and NT-4, in the murine ENS using Western blot and immunohistochemistry. The results demonstrate that enteric glial cells within the ENS express full-length TrkB at all stages tested. The ENS of TrkB deficient mice have reduced expression of glial cell markers, and a disarrangement of glial cells and the plexular neuropil. These results strongly suggest TrkB has essential roles in the normal development and maintenance of glial cells in the ENS.

The expression of ENa+C and ASIC2 proteins in Pacinian corpuscles is differently regulated by TrkB and its ligands BDNF and NT-4

Pacinian corpuscles are innervated by large myelinated Aalpha-beta axons from the large- and intermediate-sized sensory neurons of dorsal root ganglia. These neurons express different members of the degenerin/epithelial Na(+) channel (DEG/ENa(+)C) superfamily of proteins with putative mechanosensory properties, whose expression is regulated by the TrkB-BDNF system. Thus, we hypothesized that BDNF and/or NT-4 signalling through activation of TrkB may regulate the expression of molecules supposed to be necessary for the mechanosensory function of Pacinian corpuscles. To test this hypothesis we analyzed the expression and distribution of ENa(+)C subunits and acid-sensing ion channel 2 (ASIC2) in Pacinian corpuscles from 25 days old mice deficient in TrkB, BDNF and NT-4. Pacinian corpuscles in these animals are normal in number, structure, and expression of several immunohistochemical markers. Using immunohistochemistry we observed that the beta-ENa(+)C and gamma-ENa(+)C subunits, but not the alpha-ENa(+)C subunit, were expressed in wild-type animals, and they were always found in the central axon. ASIC2 immunoreactivity was found in both the central axon and the inner core cells. The absence of TrkB or BDNF abolished expression of beta-ENa(+)C and ASIC2, whereas expression of gamma-ENa(+)C did not change. Expression of beta-ENa(+)C and gamma-ENa(+)C subunits in NT-4 deficient mice was found in the axons but also in the inner core cells whereas levels of expression of ASIC2 were increased in these animals. This study suggests that expression in Pacianian corpuscles of some potential mechanosensory proteins is regulated by BDNF, NT-4 and TrkB.

Acid-sensing ion channel 2 (ASIC2) in the intestine of adult zebrafish

Acid-sensing ion channels (ASICs) in mammals monitor acid sensing and mechanoreception. They have a widespread expression in the central and peripheral nervous system, including the gut. The distribution of ASICs in zebrafish is known only in larvae and at the mRNA level. Here we have investigated the expression and cell distribution of ASIC2 in the gut of adult zebrafish using PCR, Western blot and immunohistochemistry. ASIC2 mRNA was detected in the gut, and a protein consistent with predicted ASIC2 (64kDa molecular mass) was detected by Western blot. ASIC2 positivity was found in a subpopulation of myenteric neurons in the enteric nervous system, as well in enteroendocrine epithelial cells. These data demonstrate for the first time the occurrence of ASIC2 in the gut of adult zebrafish where it presumably acts as a chemosensor and a mechanosensor.