The local mechanosensitive response of primary cardiac fibroblasts is influenced by the microenvironment mechanics.

Cardiac fibroblasts (CFs) are essential for preserving myocardial integrity and function. They can detect variations in cardiac tissue stiffness using various cellular mechanosensors, including the Ca2+ permeable mechanosensitive channel Piezo1. Nevertheless, how CFs adapt the mechanosensitive response to stiffness changes remains unclear. In this work we adopted a multimodal approach, combining the local mechanical stimulation (from 10 pN to 350 nN) with variations of culture substrate stiffness. We found that primary rat CFs cultured on stiff (GPa) substrates showed a broad Piezo1 distribution in the cell with particular accumulation at the mitochondria membrane. CFs displayed a force-dependent behavior in both calcium uptake and channel activation probability, showing a threshold at 300 nN, which involves both cytosolic and mitochondrial Ca2+ mobilization. This trend decreases as the myofibroblast phenotype within the cell population increases, following a possible Piezo1 accumulation at focal adhesion sites. In contrast, the inhibition of fibroblasts to myofibroblasts transition with soft substrates (kPa) considerably reduces both mechanically- and chemically-induced Piezo1 activation and expression. Our findings shed light on how Piezo1 function and expression are regulated by the substrate stiffness and highlight its involvement in the environment-mediated modulation of CFs mechanosensitivity.

Label-Free Analysis of Urine Samples with In-Flow Digital Holographic Microscopy.

Urinary tract infections are among the most frequent infectious diseases and require screening a great amount of urine samples from patients. However, a high percentage of samples result as negative after urine culture plate tests (CPTs), demanding a simple and fast preliminary technique to screen out the negative samples. We propose a digital holographic microscopy (DHM) method to inspect fresh urine samples flowing in a glass capillary for 3 min, recording holograms at 2 frames per second. After digital reconstruction, bacteria, white and red blood cells, epithelial cells and crystals were identified and counted, and the samples were classified as negative or positive according to clinical cutoff values. Taking the CPT as reference, we processed 180 urine samples and compared the results with those of urine flow cytometry (UFC). Using standard evaluation metrics for our screening test, we found a similar performance for DHM and UFC, indicating DHM as a suitable and fast screening technique retaining several advantages. As a benefit of DHM, the technique is label-free and does not require sample preparation. Moreover, the phase and amplitude images of the cells and other particles present in urine are digitally recorded and can serve for further investigation afterwards.

Investigation on the change of spermatozoa flagellar beating forces before and after capacitation.

The swimming forces exerted by mammalian spermatozoa during the pathway to the ovary and during the interaction with the oocyte are thought to play a fundamental role in the fertilization of the egg. In particular, a process named capacitation is of key relevance for its success. Capacitation enables spermatozoa to undergo the acrosome reaction and to exhibit different motility called hyperactivation with a change in the sperm cell tail motion from symmetric to a more asymmetric beating, characterized by wider flagellar bending at lower frequencies. Despite several studies about the mechanism that underlies capacitation, no quantitative information is available about the forces associated with sperm motility. Sperm cell motility has been widely studied with digital imaging tools and video microscopy, but these methodologies cannot provide information about the forces exerted by spermatozoa during the motion and the contribution of every single frequency of flagellar beating to the sperm cell movement. For this purpose, fluidic force microscopy was used to trap single swimming spermatozoa allowing to evaluate these parameters. We observe significant differences between capacitated and non-capacitated spermatozoa in terms of force exerted and beating frequencies. The description of the dynamics of this process is of great interest in the field of reproductive medicine. Such information could be useful to clarify unknown causes of male infertility or for the development of novel methods to assess the quality of semen samples.

Biomechanical properties of native and cultured red blood cells-Interplay of shape, structure and biomechanics.

Modern medicine increases the demand for safe blood products. Ex vivo cultured red blood cells (cRBC) are eagerly awaited as a standardized, safe source of RBC. Established culture models still lack the terminal cytoskeletal remodeling from reticulocyte to erythrocyte with changes in the biomechanical properties and interacts with membrane stiffness, viscosity of the cytoplasm and the cytoskeletal network. Comprehensive data on the biomechanical properties of cRBC are needed to take the last step towards translation into clinical use in transfusion medicine. Aim of the study was the comparative analysis of topographical and biomechanical properties of cRBC, generated from human CD34+ adult hematopoietic stem/progenitor cells, with native reticulocytes (nRET) and erythrocytes (nRBC) using cell biological and biomechanical technologies. To gain the desired all-encompassing information, a single method was unsatisfactory and only the combination of different methods could lead to the goal. Topographical information was matched with biomechanical data from optical tweezers (OT), atomic force microscopy (AFM) and digital holographic microscopy (DHM). Underlying structures were investigated in detail. Imaging, deformability and recovery time showed a high similarity between cRBC and nRBC. Young's modulus and plasticity index also confirmed this similarity. No significant differences in membrane and cytoskeletal proteins were found, while lipid deficiency resulted in spherical, vesiculated cells with impaired biomechanical functionality. The combination of techniques has proven successful and experiments underscore a close relationship between lipid content, shape and biomechanical functionality of RBC.

The Role of Cytoskeleton Revealed by Quartz Crystal Microbalance and Digital Holographic Microscopy.

The connection between cytoskeleton alterations and diseases is well known and has stimulated research on cell mechanics, aiming to develop reliable biomarkers. In this study, we present results on rheological, adhesion, and morphological properties of primary rat cardiac fibroblasts, the cytoskeleton of which was altered by treatment with cytochalasin D (Cyt-D) and nocodazole (Noc), respectively. We used two complementary techniques: quartz crystal microbalance (QCM) and digital holographic microscopy (DHM). Qualitative data on cell viscoelasticity and adhesion changes at the cell-substrate near-interface layer were obtained with QCM, while DHM allowed the measurement of morphological changes due to the cytoskeletal alterations. A rapid effect of Cyt-D was observed, leading to a reduction in cell viscosity, loss of adhesion, and cell rounding, often followed by detachment from the surface. Noc treatment, instead, induced slower but continuous variations in the rheological behavior for four hours of treatment. The higher vibrational energy dissipation reflected the cell's ability to maintain a stable attachment to the substrate, while a cytoskeletal rearrangement occurs. In fact, along with the complete disaggregation of microtubules at prolonged drug exposure, a compensatory effect of actin polymerization emerged, with increased stress fiber formation.

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.

Compromised Biomechanical Properties, Cell-Cell Adhesion and Nanotubes Communication in Cardiac Fibroblasts Carrying the Lamin A/C D192G Mutation.

Clinical effects induced by arrhythmogenic cardiomyopathy (ACM) originate from a large spectrum of genetic variations, including the missense mutation of the lamin A/C gene (LMNA), LMNA D192G. The aim of our study was to investigate the biophysical and biomechanical impact of the LMNA D192G mutation on neonatal rat ventricular fibroblasts (NRVF). The main findings in mutated NRVFs were: (i) cytoskeleton disorganization (actin and intermediate filaments); (ii) decreased elasticity of NRVFs; (iii) altered cell-cell adhesion properties, that highlighted a strong effect on cellular communication, in particular on tunneling nanotubes (TNTs). In mutant-expressing fibroblasts, these nanotubes were weakened with altered mechanical properties as shown by atomic force microscopy (AFM) and optical tweezers. These outcomes complement prior investigations on LMNA mutant cardiomyocytes and suggest that the LMNA D192G mutation impacts the biomechanical properties of both cardiomyocytes and cardiac fibroblasts. These observations could explain how this mutation influences cardiac biomechanical pathology and the severity of ACM in LMNA-cardiomyopathy.

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.

Cx32 hemichannel opening by cytosolic Ca2+ is inhibited by the R220X mutation that causes Charcot-Marie-Tooth disease.

Mutations of the GJB1 gene encoding connexin 32 (Cx32) cause the X-linked form of Charcot-Marie-Tooth disease (CMTX1), a demyelinating peripheral neuropathy for which there is no cure. A growing body of evidence indicates that ATP release through Cx32 hemichannels in Schwann cells could be critical for nerve myelination, but it is unknown if CMTX1 mutations alter the cytosolic Ca2+-dependent gating mechanism that controls Cx32 hemichannel opening and ATP release. The current study uncovered that loss of the C-terminus in Cx32 (R220X mutation), which causes a severe CMTX1 phenotype, inhibits hemichannel opening during a canonical IP3-mediated increase in cytosolic Ca2+ in HeLa cells. Interestingly, the gating function of R220X hemichannels was completely restored by both the intracellular and extracellular application of a peptide that mimics the Cx32 cytoplasmic loop. All-atom molecular dynamics simulations suggest that loss of the C-terminus in the mutant hemichannel triggers abnormal fluctuations of the cytoplasmic loop which are prevented by binding to the mimetic peptide. Experiments that stimulated R220X hemichannel opening by cell depolarization displayed reduced voltage sensitivity with respect to wild-type hemichannels which was explained by loss of subconductance states at the single channel level. Finally, experiments of intercellular diffusion mediated by wild-type or R220X gap junction channels revealed similar unitary permeabilities to ions, signalling molecules (cAMP) or larger solutes (Lucifer yellow). Taken together, our findings support the hypothesis that paracrine signalling alteration due to Cx32 hemichannel dysfunction underlies CMTX1 pathogenesis and suggest a candidate molecule for novel studies investigating a therapeutic approach.

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.

Simvastatin Rapidly and Reversibly Inhibits Insulin Secretion in Intact Single-Islet Cultures.

INTRODUCTION: Epidemiological studies suggest that statins may promote the development or exacerbation of diabetes, but whether this occurs through inhibition of insulin secretion is unclear. This lack of understanding is partly due to the cellular models used to explore this phenomenon (cell lines or pooled islets), which are non-physiologic and have limited clinical transferability. METHODS: Here, we study the effect of simvastatin on insulin secretion using single-islet cultures, an optimal compromise between biological observability and physiologic fidelity. We develop and validate a microfluidic device to study single-islet function ex vivo, which allows for switching between media of different compositions with a resolution of seconds. In parallel, fluorescence imaging provides real-time analysis of the membrane voltage potential, cytosolic Ca2+ dynamics, and insulin release during perfusion under 3 or 11 mM glucose. RESULTS: We found that simvastatin reversibly inhibits insulin secretion, even in high-glucose. This phenomenon is very rapid (<60 s), occurs without affecting Ca2+ concentrations, and is likely unrelated to cholesterol biosynthesis and protein isoprenylation, which occur on a time span of hours. CONCLUSIONS: Our data provide the first real-time live demonstration that a statin inhibits insulin secretion in intact islets and that single islets respond differently from cell lines on a short time scale. FUNDING: University of Padova, EASD Foundation.

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.

NETosis Delays Diabetic Wound Healing in Mice and Humans.

Upon activation, neutrophils undergo histone citrullination by protein arginine deiminase (PAD)4, exocytosis of chromatin and enzymes as neutrophil extracellular traps (NETs), and death. In diabetes, neutrophils are primed to release NETs and die by NETosis. Although this process is a defense against infection, NETosis can damage tissue. Therefore, we examined the effect of NETosis on the healing of diabetic foot ulcers (DFUs). Using proteomics, we found that NET components were enriched in nonhealing human DFUs. In an independent validation cohort, a high concentration of neutrophil elastase in the wound was associated with infection and a subsequent worsening of the ulcer. NET components (elastase, histones, neutrophil gelatinase-associated lipocalin, and proteinase-3) were elevated in the blood of patients with DFUs. Circulating elastase and proteinase-3 were associated with infection, and serum elastase predicted delayed healing. Neutrophils isolated from the blood of DFU patients showed an increased spontaneous NETosis but an impaired inducible NETosis. In mice, skin PAD4 activity was increased by diabetes, and FACS detection of histone citrullination, together with intravital microscopy, showed that NETosis occurred in the bed of excisional wounds. PAD4 inhibition by Cl-amidine reduced NETting neutrophils and rescued wound healing in diabetic mice. Cumulatively, these data suggest that NETosis delays DFU healing.

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.

ATP-mediated cell-cell signaling in the organ of Corti: the role of connexin channels.

UNLABELLED: Connexin 26 (Cx26) and connexin 30 (Cx30) form hemichannels that release ATP from the endolymphatic surface of cochlear supporting and epithelial cells and also form gap junction (GJ) channels that allow the concomitant intercellular diffusion of Ca(2+) mobilizing second messengers. Released ATP in turn activates G-protein coupled P2Y(2) and P2Y(4) receptors, PLC-dependent generation of IP(3), release of Ca(2+) from intracellular stores, instigating the regenerative propagation of intercellular Ca(2+) signals (ICS). The range of ICS propagation is sensitive to the concentration of extracellular divalent cations and activity of ectonucleotidases. Here, the expression patterns of Cx26 and Cx30 were characterized in postnatal cochlear tissues obtained from mice aged between P5 and P6. The expression gradient along the longitudinal axis of the cochlea, decreasing from the basal to the apical cochlear turn (CT), was more pronounced in outer sulcus (OS) cells than in inner sulcus (IS) cells. GJ-mediated dye coupling was maximal in OS cells of the basal CT, inhibited by the nonselective connexin channel blocker carbenoxolone (CBX) and absent in hair cells. Photostimulating OS cells with caged inositol (3,4,5) tri-phosphate (IP(3)) resulted in transfer of ICS in the lateral direction, from OS cells to IS cells across the hair cell region (HCR) of medial and basal CTs. ICS transfer in the opposite (medial) direction, from IS cells photostimulated with caged IP(3) to OS cells, occurred mostly in the basal CT. In addition, OS cells displayed impressive rhythmic activity with oscillations of cytosolic free Ca(2+) concentration ([Ca(2+)](i)) coordinated by the propagation of Ca(2+) wavefronts sweeping repeatedly through the same tissue area along the coiling axis of the cochlea. Oscillations evoked by uncaging IP(3) or by applying ATP differed greatly, by as much as one order of magnitude, in frequency and waveform rise time. ICS evoked by direct application of ATP propagated along convoluted cellular paths in the OS, which often branched and changed dynamically over time. Potential implications of these findings are discussed in the context of developmental regulation and cochlear pathophysiology. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11302-010-9192-9) contains supplementary material, which is available to authorized users.

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.