Calcium Signaling in the Photodamaged Skin: In Vivo Experiments and Mathematical Modeling.

The epidermis forms an essential barrier against a variety of insults. The overall goal of this study was to shed light not only on the effects of accidental epidermal injury, but also on the mechanisms that support laser skin resurfacing with intra-epidermal focal laser-induced photodamage, a widespread medical practice used to treat a range of skin conditions. To this end, we selectively photodamaged a single keratinocyte with intense, focused and pulsed laser radiation, triggering Ca2+ waves in the epidermis of live anesthetized mice with ubiquitous expression of a genetically encoded Ca2+ indicator. Waves expanded radially and rapidly, reaching up to eight orders of bystander cells that remained activated for tens of minutes, without displaying oscillations of the cytosolic free Ca2+ concentration ([Formula: see text]). By combining in vivo pharmacological dissection with mathematical modeling, we demonstrate that Ca2+ wave propagation depended primarily on the release of ATP, a prime damage-associated molecular patterns (DAMPs), from the hit cell. Increments of the [Formula: see text] in bystander cells were chiefly due to Ca2+ release from the endoplasmic reticulum (ER), downstream of ATP binding to P2Y purinoceptors. ATP-dependent ATP release though connexin hemichannels (HCs) affected wave propagation at larger distances, where the extracellular ATP concentration was reduced by the combined effect of passive diffusion and hydrolysis due to the action of ectonucleotidases, whereas pannexin channels had no role. Bifurcation analysis suggests basal keratinocytes have too few P2Y receptors (P2YRs) and/or phospholipase C (PLC) to transduce elevated extracellular ATP levels into inositol trisphosphate (IP3) production rates sufficiently large to sustain [Formula: see text] oscillations.

Biomechanics of Ex Vivo-Generated Red Blood Cells Investigated by Optical Tweezers and Digital Holographic Microscopy.

Ex vivo-generated red blood cells are a promising resource for future safe blood products, manufactured independently of voluntary blood donations. The physiological process of terminal maturation from spheroid reticulocytes to biconcave erythrocytes has not been accomplished yet. A better biomechanical characterization of cultured red blood cells (cRBCs) will be of utmost interest for manufacturer approval and therapeutic application. Here, we introduce a novel optical tweezer (OT) approach to measure the deformation and elasticity of single cells trapped away from the coverslip. To investigate membrane properties dependent on membrane lipid content, two culture conditions of cRBCs were investigated, cRBCPlasma with plasma and cRBCHPL supplemented with human platelet lysate. Biomechanical characterization of cells under optical forces proves the similar features of native RBCs and cRBCHPL, and different characteristics for cRBCPlasma. To confirm these results, we also applied a second technique, digital holographic microscopy (DHM), for cells laid on the surface. OT and DHM provided related results in terms of cell deformation and membrane fluctuations, allowing a reliable discrimination between cultured and native red blood cells. The two techniques are compared and discussed in terms of application and complementarity.

Mouse Panx1 Is Dispensable for Hearing Acquisition and Auditory Function.

Panx1 forms plasma membrane channels in brain and several other organs, including the inner ear. Biophysical properties, activation mechanisms and modulators of Panx1 channels have been characterized in detail, however the impact of Panx1 on auditory function is unclear due to conflicts in published results. To address this issue, hearing performance and cochlear function of the Panx1-/- mouse strain, the first with a reported global ablation of Panx1, were scrutinized. Male and female homozygous (Panx1-/-), hemizygous (Panx1+/-) and their wild type (WT) siblings (Panx1+/+) were used for this study. Successful ablation of Panx1 was confirmed by RT-PCR and Western immunoblotting in the cochlea and brain of Panx1-/- mice. Furthermore, a previously validated Panx1-selective antibody revealed strong immunoreactivity in WT but not in Panx1-/- cochleae. Hearing sensitivity, outer hair cell-based "cochlear amplifier" and cochlear nerve function, analyzed by auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) recordings, were normal in Panx1+/- and Panx1-/- mice. In addition, we determined that global deletion of Panx1 impacts neither on connexin expression, nor on gap-junction coupling in the developing organ of Corti. Finally, spontaneous intercellular Ca2+ signal (ICS) activity in organotypic cochlear cultures, which is key to postnatal development of the organ of Corti and essential for hearing acquisition, was not affected by Panx1 ablation. Therefore, our results provide strong evidence that, in mice, Panx1 is dispensable for hearing acquisition and auditory function.

Design and Construction of a Cost-Effective Spinning Disk System for Live Imaging of Inner Ear Tissue.

Confocal imaging of fluorescent probes offers a powerful, non-invasive tool which enables data collection from vast population of cells at high spatial and temporal resolution. Spinning disk confocal microscopy parallelizes the imaging process permitting the study of dynamic events in populations of living cells on the millisecond time scale. Several spinning disk microscopy solutions are commercially available, however these are often poorly configurable and relatively expensive. This chapter describes a procedure to assemble a cost-effective homemade spinning disk system for fluorescence microscopy, which is highly flexible and easily configurable. We finally illustrate a reliable protocol to obtain high-quality Ca(2+) and voltage imaging data from cochlear preparations.

Reduced phosphatidylinositol 4,5-bisphosphate synthesis impairs inner ear Ca2+ signaling and high-frequency hearing acquisition.

Phosphatidylinositol phosphate kinase type 1γ (PIPKIγ) is a key enzyme in the generation of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] and is expressed at high levels in the nervous system. Homozygous knockout mice lacking this enzyme die postnatally within 24 h, whereas PIPKIγ(+/-) siblings breed normally and have no reported phenotype. Here we show that adult PIPKIγ(+/-) mice have dramatically elevated hearing thresholds for high-frequency sounds. During the first postnatal week we observed a reduction of ATP-dependent Ca(2+) signaling activity in cochlear nonsensory cells. Because Ca(2+) signaling under these conditions depends on inositol-1,4,5-trisphosphate generation from phospholipase C (PLC)-dependent hydrolysis of PI(4,5)P(2), we conclude that (i) PIPKIγ is primarily responsible for the synthesis of the receptor-regulated PLC-sensitive PI(4,5)P(2) pool in the cell syncytia that supports auditory hair cells; (ii) spatially graded impairment of this signaling pathway in cochlear nonsensory cells causes a selective alteration in the acquisition of hearing in PIPKIγ(+/-) mice. This mouse model also suggests that PIPKIγ may determine the level of gap junction contribution to cochlear development.

Multiphoton fluorescence microscopy with GRIN objective aberration correction by low order adaptive optics.

Graded Index (GRIN) rod microlenses are increasingly employed in the assembly of optical probes for microendoscopy applications. Confocal, two-photon and optical coherence tomography (OCT) based on GRIN optical probes permit in-vivo imaging with penetration depths into tissue up to the centimeter range. However, insertion of the probe can be complicated by the need of several alignment and focusing mechanisms along the optical path. Furthermore, resolution values are generally not limited by diffraction, but rather by optical aberrations within the endoscope probe and feeding optics. Here we describe a multiphoton confocal fluorescence imaging system equipped with a compact objective that incorporates a GRIN probe and requires no adjustment mechanisms. We minimized the effects of aberrations with optical compensation provided by a low-order electrostatic membrane mirror (EMM) inserted in the optical path of the confocal architecture, resulting in greatly enhanced image quality.

Purinergic signalling and intercellular Ca2+ wave propagation in the organ of Corti.

Extracellular ATP is a key neuromodulator of visual and auditory sensory epithelia. In the rat cochlea, pharmacological dissection indicates that ATP, acting through a highly sensitive purinergic/IP(3)-mediated signaling pathway with (little or) no involvement of ryanodine receptors, is the principal paracrine mediator implicated in the propagation of calcium waves through supporting and epithelial cells. Measurement of sensitivity to UTP and other purinergic agonists implicate P2Y(2) and P2Y(4) as the main P2Y receptor isoforms involved in these responses. Ca2+ waves, elicited under highly reproducible conditions by carefully controlling dose (1 microM) and timing of focal agonist application (0.2s), extended over radial distance greater than 160 microm from the source, identical to those activated by damaging single outer hair cells. Altogether, these results indicate that intercellular calcium waves are a robust phenomenon that confers a significant ability for cell-cell communication in the mammalian cochlea. Further ongoing research will reveal the roles that such Ca2+ waves play in the inner ear.

A mechanism for sensing noise damage in the inner ear.

Our sense of hearing requires functional sensory hair cells. Throughout life those hair cells are subjected to various traumas, the most common being loud sound. The primary effect of acoustic trauma is manifested as damage to the delicate mechanosensory apparatus of the hair cell stereocilia. This may eventually lead to hair cell death and irreversible deafness. Little is known about the way in which noxious sound stimuli affect individual cellular components of the auditory sensory epithelium. However, studies in different types of cell cultures have shown that damage and mechanical stimulation can activate changes in intracellular free calcium concentration ([Ca(2+)](i)) and elicit intercellular Ca(2+) waves. Thus an attractive hypothesis is that changes in [Ca(2+)](i), propagating as a wave through support cells in the organ of Corti, may constitute a fundamental mechanism to signal the occurrence of hair cell damage. The mechanism we describe here exhibits nanomolar sensitivity to extracellular ATP, involves regenerative propagation of intercellular calcium waves due to ATP originating from hair cells, and depends on functional IP(3)-sensitive intracellular stores in support cells.

Presynaptic calcium stores modulate afferent release in vestibular hair cells.

Hair cells, the mechanoreceptors of the acoustic and vestibular system, are presynaptic to primary afferent neurons of the eighth nerve and excite neural activity by the release of glutamate. In the present work, the role played by intracellular Ca2+ stores in afferent transmission was investigated, at the presynaptic level, by monitoring changes in the intracellular Ca2+ concentration ([Ca2+]i) in vestibular hair cells, and, at the postsynaptic level, by recording from single posterior canal afferent fibers. Application of 1-10 mm caffeine to hair cells potentiated Ca2+ responses evoked by depolarization at selected Ca2+ hot spots, and also induced a graded increase in cell membrane capacitance (DeltaCm), signaling exocytosis of the transmitter. Ca2+ signals evoked by caffeine peaked in a region located approximately 10 microm from the base of the hair cell. [Ca2+]i increases, similarly localized, were observed after 500 msec depolarizations, but not with 50 msec depolarizations, suggesting the occurrence of calcium-induced calcium release (CICR) from the same stores. Both Ca2+ and DeltaCm responses were inhibited after incubation with ryanodine (40 microm) for 8-10 min. Consistent with these results, afferent transmission was potentiated by caffeine and inhibited by ryanodine both at the level of action potentials and of miniature EPSPs (mEPSPs). Neither caffeine nor ryanodine affected the shape and amplitude of mEPSPs, indicating that both drugs acted at the presynaptic level. These results strongly suggest that endogenous modulators of the CICR process will affect afferent activity elicited by mechanical stimuli in the physiological frequency range.

Permeability and gating properties of human connexins 26 and 30 expressed in HeLa cells.

Human connexins 26 and 30 were expressed either through the bicistronic pIRES-EGFP expression vector or as EYFP-tagged chimeras. When transiently transfected in communication-incompetent HeLa cells, hCx26-pIRES transfectants were permeable to dyes up to 622 Da, but were significantly less permeable to 759 Da molecules. Under the same conditions, permeability of hCx26-EYFP fusion products was comparable to that of hCx26-pIRES, but with significant increase in diffusion at 759 Da, possibly as a consequence of having selected large fluorescent junctional plaques. Dye transfer was limited to 457 Da in hCx30-EYFP transfectants. When reconstructed from confocal serial sections, fluorescent plaques formed by hCx26-EYFP and hCx30-EYFP appeared irregular, often with long protrusions or deep invagination. Similar plaques were observed following immunostaining both in cells transfected with hCx26-pIRES and in HeLa cells stably transfected with mouse Cx26. Tissue conductance (Tg(j)) displayed significantly smaller values (28.8+/-1.8 nS) for stably transfected mCx26 than transiently transfected hCx26 (43.5+/-3.3 nS). These differences reflected in distinct functional dependence of normalized junctional conductance (G(j)) on transjunctional voltage (V(j)). The half-activation voltage for G(j) was close to +/-95 and +/-58 mV in mCx26 and hCx26, respectively. The corresponding parameters for hCx30 transfectants were Tg(j)= 45.2 +/- 3.5 nS and V(0)= +/- 34 mV. These results highlight unexpected differences between mCx26 and hCx26 in this expression system, reinforce the concept that channel permeability may be related to Cx level expression, and indicate that fusion of hCx30 to GFP colour mutants produces channels that are suitable for permeability and gating studies.