A key property of the human cornea is to maintain its curvature and consequently its refraction capability despite daily changes in intraocular pressure. This is closely related to the multiscale structure of the corneal stroma, which consists of 1–3 µm-thick stacked lamellae made of thin collagen fibrils. Nevertheless, the distribution, size, and orientation of these lamellae along the depth of the cornea are poorly characterized up to now. In this study, we use second harmonic generation (SHG) microscopy to visualize the collagen distribution over the full depth of 10 intact and unstained human corneas (500–600 µm thick). We take advantage of the small coherence length in epi-detection to axially resolve the lamellae while maintaining the corneal physiological curvature. Moreover, as raw epi-detected SHG images are spatially homogenous because of the sub-wavelength size of stromal collagen fibrils, we use a polarimetric approach to measure the collagen orientation in every voxel. After a careful validation of this approach, we show that the collagen lamellae (i) are mostly oriented along the inferior–superior axis in the anterior stroma and along the nasal-temporal axis in the posterior stroma, with a gradual shift in between and (ii) exhibit more disorder in the anterior stroma. These results represent the first quantitative characterization of the lamellar structure of the human cornea continuously along its entire thickness with micrometric resolution. It also shows the unique potential of P-SHG microscopy for imaging of collagen distribution in thick dense tissues.  Its mechanical properties are also crucial for maintaining the same curvature and the subsequent refraction capability despite daily changes in intraocular pressure and external shocks. All these properties are closely related to the structure of the stroma, the main layer of the cornea2. The stroma is around 500 µm thick in the cornea center and consists of 1–3 µm-thick stacked lamellae made of collagen fibrils (around 30 nm in diameter) aligned and regularly packed to ensure cornea transparency1. These lamellae are roughly organized parallel to the cornea surface, with different collagen orientations in sequential lamellae. However, the distribution of these lamellae as well as their size and orientation to the cornea surface vary along the depth of the cornea. For instance, in the anterior part of the stroma, lamellae are interwoven and collagen fibrils can run from one lamella to the next one, while lamellae seem to be more regularly stacked in the posterior stroma3. Altogether, the anisotropic hierarchical organization of the cornea and its physiological consequences on cornea biomechanics have not been accurately characterized yet because of the limitations of conventional techniques.The cornea is the most anterior segment of the eye and is characterized by its transparency and refractive power5. However, they are two-dimensional imaging techniques with a limited field of view, which is not suitable for a precise characterization of 500 µm-thick tissue around 10 mm in diameter. Optical coherence tomography (OCT) and reflectance confocal microscopy are three-dimensional (3D) imaging techniques that are routinely used in clinical ophthalmology, but they can hardly resolve collagen lamellae and cannot visualize their orientation6. X-ray scattering has been used successfully to measure the average orientation of collagen lamellae over the full thickness of the cornea7. It has shown that collagen lamellae are mainly oriented along the nasal–temporal and inferior–superior axes in the center of the cornea, while an orientation tangential to the circumference predominates in the periphery7. Moreover, micro-focus X-ray scattering on thick transverse sections of corneas has shown a difference in the inclination of these lamellae from the center to the periphery and through the depth8. However, this technique cannot resolve the in-plane collagen orientation within lamellae through the depth of the stroma and has limited transverse resolution.Transmission and scanning electron microscopies are highly powerful imaging methods that can resolve the collagen fibrils within the lamellae and show the steep changes of collagen orientation between adjacent lamellae along the corneal depth10. This multiphoton modality has already been used in human corneas in combination with two-photon excited fluorescence (2PEF) to visualize keratocytes, which are cells within the stroma21. However, usual epi-detected SHG images with no polarization resolution are homogeneous, except for low signal lines related to stromal striae that correspond to undulations of lamellae and may have a mechanical role to absorb increases in intraocular pressure and external shocks22. On the other hand, trans-detected SHG images exhibit a longer coherence length, which can encompass collagen fibrils with opposite polarities, and thus results in striated patterns oriented along the collagen fibrils. Unfortunately, collagen orientation is not processed efficiently in the numerous voxels showing cross striations. Moreover, trans-detection is not compatible with usual corneal chambers that reproduce physiological conditions2. Accordingly, no complete multiscale structure of the human cornea has been reported up to now.Second harmonic generation (SHG) microscopy is nowadays the gold standard technique for in situ visualization of collagen 3D organization in unstained biological tissues26 and shown that epi-detection of P-SHG signals is an efficient and precise method to visualize the collagen orientation over the whole depth of human corneas (500–600 µm). We have optimized our P-SHG imaging protocols to get reliable P-SHG data in depth and implemented a data-processing workflow to estimate automatically the mean collagen orientation in every voxel as well as the precision of this measure. Using this approach, we have obtained 3D reconstructions of collagen orientation over the full depth of 10 whole human corneas as well as the lamellae orientation distribution along the depth of the cornea.In this study, we have implemented polarization-resolved SHG (P-SHG) microscopyφ of collagen fibrils and (iii) the map of the coefficient of determination R2.We measured the collagen orientation in the center of 10 human corneas using P-SHG microscopy. Figure 16. Conversely, the small coherence length in the backward direction results in the signal build-up on a smaller scale (≈100 nm) and almost homogeneous images, except for long areas with no signal. These structures can be attributed either to stromal striae or to keratocytes. Transverse reconstructions of the epi-detected <SHG> images  and correspond either to stromal striae (white arrowheads) or keratocytes where the SHG signal is too low or to crossings of lamellae (yellow arrows) as observed in the trans-detected <SHG> image. In the latter case, the focal volume contains fibrils with different orientations, so that the overall cylindrical symmetry does not apply and Eqs. , then along Y-axis  and finally again along X-axis (P-SHG-3). Figure 27; (ii) slight optical heterogeneities of the cornea, inducing mild depolarization and birefringence.As P-SHG is sensitive to polarization distortionsxis Fig.  is firstis P-SHG-. Figure We have quantified this artifact as a function of the imaging depth within the cornea by calculating the normalized distance  and processed the mean angular difference between the two maps at every depth using Eq.  numerical rotation by 90° of the orientation distributions extracted from the second P-SHG stack and averaging with the distributions extracted from the first P-SHG stack . Nevertheless, Fig. ck Movie . This prion Fig.  must thein fine the orientation distribution of stromal lamellae. Figure Following this approach based on two sequential P-SHG acquisitions for two perpendicular orientations of the cornea, we obtain collagen orientation distributions that represent R2. This parameter is close to 1, which indicates that the orientation distributions are well described by the sum of two Van Mises distributions and an isotropic component. The angular shift Δμ between the two peaks is 86°  for the orientation distribution over the whole cornea thickness (resp. in the 2nd and 3rd parts), which is very close to 90°. This is fully consistent with the usual description of the human cornea as a stacking of collagen lamellae with two main perpendicular orientations along the nasal-temporal and inferior–superior axes. However, our results show that this usual description must be reconsidered in the anterior stroma: the two peaks are not well-defined in all corneas, so the average distribution show wide peaks  that are not exactly perpendicular (Δμ = 74°). Moreover, our data highlight variations in the lamellae distribution along all the stroma. First, the peaks become thinner and thinner from the anterior to the posterior stroma, as quantified by the continuous increase in the κ parameters. It means that the lamellae are better aligned along two perpendicular axes in the posterior stroma than in the middle one and even less in the anterior one. Second, the main orientation of the lamellae shifts gradually from the inferior–superior axis in the anterior stroma to the nasal–temporal axis in the posterior stroma, as quantified by the increase in the cNT parameter and the concomitant decrease in the cIS parameter. This feature is not visible in the full-thickness distribution where the variations are averaged, which results in well-balanced peaks with a similar percentage of lamellae along the two perpendicular axes. The same averaging applies to all techniques with no 3D resolution, notably to X-rays scattering data that are recorded on the full thickness of the cornea. Accordingly, the orientation distribution obtained by P-SHG on the full thickness of the cornea is in good agreement with the one obtained by X-ray scattering by Meek et al32. Note however that the isotropic component is quite strong in X-ray data, while it is minor in P-SHG data, as already reported33. This effect is nevertheless difficult to quantify because the isotropic coefficient in Eq.  use a custom-designed cornea holder that maintains physiological conditions, mainly the cornea curvature, and minimize polarization artifacts due to tissue deformation; (ii) carefully align the microscope setup and optimize the linearity of the excitation polarization, in order to maximize the contrast of the polarimetric diagram in every voxel; (iii) take advantage of epi-detection to record SHG images with low coherence length and maximize the number of voxels encompassing unique lamellae, with well-defined collagen orientation, rather than two lamellae with two different orientations; (iv) implement an automatic image-processing workflow to calculate the collagen orientation in every voxel from the P-SHG images and eliminate unreliable data based on 35. As the cornea is transparent and exhibits globally a very low birefringence, we expect negligible issues due to scattering or birefringence, but the polarimetric diagrams may be slightly distorted in-depth due to slight misalignment or tissue heterogeneities. Most importantly, the focal volume can encompass two sequential lamellae composed of collagen with different orientations, which invalidates the cylindrical symmetry hypothesis used to analyze P-SHG data36. This effect is much lower in epi-detection, because of the much smaller coherence length, but it is still present in a few voxels. We have therefore filtered out the voxels where the polarimetric data do not fit the theoretical Eq. 39. Application of an R2 threshold is anyway easy to implement and efficient to eliminate unreliable P-SHG data40.These key points are all related to the sensitivity of polarimetric measurements to any optical defect. Accordingly, P-SHG microscopy has been reported to be prone to many artifacts, especially in depth0.7 Fig. . We may Beyond this careful filtering of the P-SHG data, we have also validated these orientation measurements by two complementary methods. This is mandatory because FFT processing of P-SHG data always provides an orientation, whatever the matching of the polarimetric response to the theory. First, we have verified that the orientation distributions obtained by P-SHG rotate when the cornea is physically rotated under the objective lens, which advantageously requires no ground truth. Second, we have compared in every voxel the orientations obtained by epi-detected P-SHG and by processing the trans-detected <SHG> images using OrientationJ, considered as the ground truth. Both methods are consistent in indicating that epi-detected P-SHG measurements of collagen orientation in the cornea are valid until approximately 350 µm with about 4° angular precision. Nevertheless, collagen orientation distributions can be measured deeper in a reliable way by averaging a series of measurements performed with two perpendicular orientations of the cornea. This practical protocol provides quantitative measurements of the lamellae orientation distribution along the full thickness of the cornea which is up to 600 µm.33. This is not possible using image processing of trans-detected <SHG> data because of the larger coherence length, as already noted41. Alternatively, the use of polarization-resolved third harmonic generation (THG) microscopy43 may be explored, but certainly at the expense of smaller signals that would require longer acquisition times, and only using trans-detection.We have used this optimized protocol to visualize and quantify the lamellar structure of 10 entire human corneas. We have used a large field of view encompassing few lamellae (250 µm) and a small voxel size enabling lamellar resolution in the axial direction  in order to better visualize the lamellar domains than in previous reports15. Compared to previous studies, our data further show the evolution of the lamellae size and orientation in 3D all along the stroma in the very same cornea. As a perspective, these lamellar domains may be individually segmented and quantified. It would nevertheless require to implement new approaches as established segmentation methods do not apply to orientation data that are circular data (0° = 360°), while our quantitative measures of the collagen orientation distributions are already highly informative. They consistently show in all the corneas under study that the lamellae are mostly aligned along the nasal-temporal and the inferior-superior directions, in agreement with the literature32. They further provide new quantitative information about the structural variations along the corneal depth that cannot be obtained by other techniques: first, the lamellae better align along two perpendicular axes in the posterior stroma than in the anterior one, which shows a more disordered distribution. Second, the collagen lamellae are mostly oriented along the inferior–superior axis in the anterior stroma and along the nasal–temporal axis in the posterior stroma, with a gradual shift in between.3D reconstructions of P-SHG data thus show in a unique way the stacking of collagen lamellae with different orientations all along the thickness of the cornea. It confirms that lamellar domains are larger and better-aligned parallel to the corneal surface in the posterior stroma than in the anterior one44. Cornea mechanical properties are routinely estimated using air-puff measurements in vivo, which does not provide extensive mechanical data, and more complete ex vivo measurements are still challenging46. Numerical models are thus mandatory for the prediction of the cornea mechanical properties and refraction capability, but only recent studies have taken into account the lamellar structure of the cornea, still in a simplified manner because of the lack of accurate orientation data48. This may be overcome by using our depth-resolved lamellae orientation distribution obtained from P-SHG, whereas such depth-resolved information with appropriate resolution and accuracy cannot be obtained using more conventional techniques such as OCT, X-ray scattering, or electron microscopy. This may then enable improved numerical simulation of the mechanical behavior of healthy corneas as well as visually impaired ones. The most direct application would be to provide a better patient-specific prediction of stromal remodeling after refractive surgery and consequently improve the correction accuracy of this surgery. P-SHG may also be used to measure the lamellar structure of keratoconic corneas. Keratoconus is a corneal pathology of increasing prevalence that is characterized by gradual corneal thinning and in the most advanced cases, by the formation of a cone that severely impairs vision. Keratoconus is also characterized by poor mechanical properties (low stiffness), which are associated with a degradation of the stroma structure45. However, no extensive full-depth characterization of the lamellar structure of keratoconic cornea has been reported up to now. P-SHG imaging of keratoconic corneal buttons may bridge this gap and provide crucial data enabling numerical simulations of the mechanical properties of keratoconic cornea48. It would be crucial for the understanding of stromal remodeling during keratoconus and it may possibly improve its early diagnosis.These structural data are crucial for the understanding of the cornea function. The refraction capability of the cornea is indeed directly linked to its curvature, which must therefore be kept constant despite daily variations in the intraocular pressure. This is guaranteed by highly specific mechanical properties that are closely connected to the lamellar structure of the stromaIn conclusion, we have taken advantage of the small coherence length in epi-detected SHG microscopy and of P-SHG sensitivity to collagen orientation in order to map the lamellar structure of human corneas with micrometric resolution. We have carefully assessed the precision of our orientation measurements and obtained 3D reconstructions in the center of 10 entire corneas over their full thickness. Quantitative analyses of these data show in a unique way the evolution of the lamellar structure along the depth of the cornea. A key advantage of our method is to use epi-detection and consequently to be compatible with any corneal chamber or with in vivo imaging. It could therefore be used to record stromal remodeling during inflation assays in ex vivo corneas, in order to measure advanced mechanical properties of cornea. Our approach is readily applicable to any tissue and shows the unique potential of epi-detected P-SHG microscopy for imaging thick collagen-rich tissues.This study was carried out according to the tenets of the Declaration of Helsinki and followed international ethical requirements for human tissues. Handling of human corneas was declared to the French administration (CODECOH agreement DC-2018-3300). Ten human corneas were obtained from the French Eye Bank , which were unsuitable for transplantation and authorized for scientific use by the donor family. These corneas were preserved in an appropriate culture medium  in an incubator set at 31°. They were then placed for 48 h in Stemalpha 3 without phenol red to induce deswelling and elimination of the phenol red present in the previous culture medium, prior to fixation in 4% paraformaldehyde (PFA) for 24 h at 4 °C. Fixed corneas were stored in 1% PFA at 4 °C until imaging. Physiological data, storage durations, and thicknesses of all corneas are given in Supplementary Table X or Y axis in the imaging plane.A specific cornea holder was designed for P-SHG imaging in order to comply with two important requirements: (i) maintain the physiological curvature of the cornea as in usual corneal chambers since we observed polarization artifacts due to diattenuation in pieces of flattened corneas stuck between two coverslips during preliminary P-SHG experiments; (ii) enable trans-detection of SHG signals for validation purpose of epi-detected orientation maps (see below), which is not possible in usual corneal chambers. We used a small plano-convex lens with the same diameter and radius of curvature as the human cornea . The cornea was placed on this lens, in turn, placed on a coverslip, and the sclera was clamped with a plastic ring maintained by a metallic screwed holder Fig. . Ophthal21  objective lens with water immersion  was used with nominal resolutions (FWHM) of 0.35 µm  × 1.2 µm  and a field of view of 540 × 540 µm2. As the cornea refractive index (≈1.37) is different from the one of water, the objective collar was adjusted before each acquisition in order to minimize spherical aberrations and avoid low contrast in depth. It was set to the position that maximized the SHG signal at half the depth of every cornea  at the expense of a slightly degraded resolution at the top and bottom of the sample, which was measured as 0.8 µm  × 2.6 µm . SHG signals were detected in the epi- and trans-directions by means of photon-counting photomultiplier tubes  and suitable spectral filters to reject the excitation beam and select the second harmonic signal at 430 nm . Excitation power was linearly increased along the depth of the cornea to compensate for signal decrease, starting from 40 to 55 mW at the epithelium side and reaching 60–100 mW when imaging the endothelium side.SHG images were recorded by use of a custom-built upright laser scanning multiphoton microscope as previously described21 Fig. . Excitat2. Series of 18 images were acquired every 10° between 0° and 170° using 1 µm pixel size and 5 µs pixel dwell time. Stacks of such P-SHG images were acquired along the full depth of the corneas with 1 µm axial steps, resulting in 2–3 h acquisition per cornea.P-SHG imaging was performed by recording a series of SHG images excited by linearly polarized incident electric fields with different orientations Fig. . The oriχ(2), which links the nonlinear polarization Pω2 to the excitation electric field at fundamental frequency Eω49:i, j, k stand for x, y, z. Considering that the collagen fibrils exhibit a cylindrical symmetry and that the Kleinman symmetry is valid, there are only 2 independent components in the fibril frame (xyz): χxxx and x is the fibril axis. The P-SHG signal intensity from a collagen fibril oriented at an angle φ to the X-axis in the image plane XY then depends on the orientation angle θ to the X-axis of the linearly polarized excitation field . The parameter K includes experimental geometrical parameters and the square of the excitation laser intensity.The susceptibility components a0, a2, and a4 which are linear combinations of the susceptibility components of the collagen fibrils23:Equation  can be wφ in every pixel . The theoretical angular precision is about 1° after the application of a 6 × 6 averaging filter to increase the signal-to-noise ratio30. Our data-processing workflow also provides21: (i) the SHG signal averaged over all linear polarizations: R2 that compares the experimental data and the curve obtained from the FFT parameters in every pixel, in order to validate the orientation measure  indicates the collagen orientation φ and the brightness (V) the R2 parameter.We thus use an FFT algorithm to process our data and determine the orientation ure Fig. . Angles φ in the range , not the polarity and that polar plots are therefore extended to φ + π.Orientation maps obtained from P-SHG data provide the distribution of the collagen orientation in the field of view. These distributions are computed using 1° angular bins either at every depth or in specific regions of the stroma by merging the orientation data over a specific range of depth. Notably, distributions are computed over the full stromal thickness or by dividing the stromal thickness in 3 thirds that correspond approximately to anterior, middle, and posterior stromal regions. The resulting distributions are normalized to the total number of valid pixels and depicted as polar plots. Note that P-SHG provides only the orientation p(φ) are finally fitted by the sum of two Van Mises distributions48:C(κ) is a normalization constant so that μ and 1/κ are analog to the average and the variance of a normal distribution. The angles are doubled in the exponential because φ covers only half the full range of directions . This fitting is applied to the average of the 2 distributions obtained for 2 perpendicular orientations of the cornea, after angular registration, as explained below.These orientation distributions z for orientation Oi of the cornea. The minimum Za. It is normalized to the maximum distance between 2 normalized distributions (i.e. to 2). This distance is also calculated on the orientation distributions computed over the 3 thirds of the stroma and over its full thickness.In order to verify the validity of these orientation distributions, a series of P-SHG image stacks are acquired and processed for different orientations of every cornea. The orientation distributions, but the first one, are initially angularly shifted to correspond to the same cornea orientation as in the first acquisition. The registration angle is calculated by minimizing the distance of the distributions in the anterior region. This distance between two distributions is calculated as the summation of the absolute difference between the distribution values at each angle Fig. . After r28. We use a window size of 6 pixels and filter out the pixels where the coherency is smaller than 0.7. We then compare the orientation maps obtained by the two methods in every image of the z-stack by computing the average of their pixel-by-pixel absolute difference:Z.As trans-detected <SHG> images show striations reflecting the orientation of the collagen fibrils, we also determine the orientation distribution by image processing of Supplementary InformationMovie 1. P-SHG imaging of a human cornea (Cornea n°1 in table S1) over its full thicknessMovie 2. Protocol to obtain reliable orientation distribution of collagen in the posterior stroma


Using a high-speed optical trap to examine dimeric PCDH15, we find that the protein’s configuration is sensitive to Ca2+ and that the molecule exhibits limited unfolding at a physiological Ca2+ concentration. PCDH15 can therefore modulate its stiffness without undergoing large unfolding events in physiological Ca2+ conditions. The experimentally determined stiffness of PCDH15 accords with published values for the stiffness of the gating spring, the mechanical element that controls the opening of mechanotransduction channels. When PCDH15 has a point mutation, V507D, associated with non-syndromic hearing loss, unfolding events occur more frequently under tension and refolding events occur less often than in the wild-type protein. Our results suggest that the maintenance of appropriate tension in the gating spring is critical to the appropriate transmission of force to transduction channels, and hence to hearing.Mechanical force controls the opening and closing of mechanosensitive ion channels atop the hair bundles of the inner ear. The filamentous tip link connecting transduction channels to the tallest neighboring stereocilium modulates the force transmitted to the channels and thus changes their probability of opening. Each tip link comprises four molecules: a dimer of protocadherin 15 and a dimer of cadherin 23, all of which are stabilized by Ca When the hair bundle is deflected towards its tall edge by a sound stimulus, the stereocilia pivot and tense the gating springs that control the opening of the mechanoelectrical transduction channels atop each stereocilium. When sufficient tension has been conveyed, the channels open and allow cations within the endolymphatic fluid surrounding the hair bundles to flow into the cells and transduction to occur2  and a dimer of cadherin 23 (CDH23). Because abolishing the tip links halts mechanotransduction4 whereas allowing the tip links to regenerate restores sensitivity5, the tip link—or the attachments at its two ends—likely controls the opening and closing of the channels and serves as the gating spring. Moreover, the hundreds of mutations in tip-link proteins that result in human hearing loss10 underscore the role of the tip link in hearing.It is plausible that the gating springs are the filamentous tip links that connect the transduction channels of a stereocilium to its tallest neighbor. Each tip link comprises four protein molecules11 implicates the protein in channel gating. PCDH15 comprises 11 extracellular cadherin (EC) domains and a protocadherin 15-interacting, channel-associated (PICA) domain, also known as MAD1212 or EL11 d of EC5 , which iaccurate . The preaccurate ,C. This accurate . Disrupt2+ decreased from saturating to absent, PCDH15 V507D exhibited a striking pattern of increasing dysregulation. At a high Ca2+ concentration of 3 mM, and in contradistinction to the wild-type dimer, there were two populations of V507D molecules. Some V507D molecules appeared to behave more like wild-type PCDH15 at the same saturating level of Ca2+, with the minimal unfolding observed in individual cycles reflected by the single bright branch on the heatmap , a distance consistent with the range of unfolding expected from the inter-domain linker regions. Because the difference between states 1 and 2 is smaller than what we would expect for unfolding of an entire EC cadherin, we call states 1 and 2 the folded states—that is, the states in which all EC domains and the PICA domain apparently remain folded. The differences between states 2 and 3 and between states 3 and 4 are respectively 29.9 ± 0.7 nm  and 30.0 ± 1.3 nm , values consistent with the expectation for unfolding of a complete EC or PICA domain. The difference between states 4 and 5 is 17.6 ± 2.0 nm , a value that does not accord with unfolding of a whole domain but might represent partial unfolding of a domain. Finally, the difference between states 5 and 6 is 72.2 ± 6.0 nm , a value similar to that expected for the unfolding of two entire EC or PICA domains.To understand PCDH15 in various concentrations of Caf PCDH15 . The defs states . Notwith2+ concentration and V507D mutation on how PCDH15 traversed the accessible state space, we segmented a molecule’s trajectory during each cycle by the conformational changes that occurred and assigned each segment to a state through k-nearest-neighbor classification  of the instances. In the absence of Ca2+, however, PCDH15 refolded back to state 1 or 2 only 62.9 ± 10.5 % (N = 6 datasets) of the time  of the occasions. At a physiological level of Ca2+ this value decreased to 59.3 ± 8.0 % (N = 16 datasets) in stark contrast with the wild-type protein, for which refolding to state 1 or 2 occurred in most instances at the same concentration of Ca2+. Finally, in the absence of Ca2+, PCDH15 V507D refolded to state 1 or state 2 in only 24.0 ± 16.2 % (N = 4 datasets) of the cases, which signified a severe reduction in refolding ability compared to the native protein. The differences in refolding rates between wild-type and V507D dimers suggest that the point mutation disrupts the refolding ability of PCDH15. V507D dimers unfolded more often than wild-type dimers and refolded properly less often.In the V507D mutant at a saturating level of Ca2+ condition. In 587 determinations at physiological Ca2+ levels, PCDH15 had an enthalpic stiffness of 6.4 ± 0.4 mN×m−1. If we assume that CDH23, which is approximately 2.3 times as long as PCDH15, has mechanical properties similar to those of PCDH15, its stiffness would be about 2.8 mN×m−1. For both proteins in series, the stiffness −1. Measurements have shown the stiffness of the gating spring to be between 0.5 mN×m−1 and 4 mN×m−1, depending on the characteristic frequency along the cochlea15, so our value for the enthalpic stiffness of PCDH15 lies within the expected stiffness range of the gating spring. Furthermore, the entropic stiffness of each state is less than the calculated enthalpic stiffness (We calculated the enthalpic stiffness for PCDH15 and PCDH15 V507D by finding the inverse spatial derivative of each cycle in the high force regime—above 30 pN, the force at which the relationship between force and extension became essentially linear—and averaging the values across all data for each Catiffness , which i2+ concentration and over the relevant force range, unfolding of entire EC domains is not the primary response of PCDH15 to force. Instead, the response likely comprises a series of smaller unfolding events that stem from the unfolding of the inter-domain linkers and possibly parts of EC domains.Our findings indicate that PCDH15, in the dimeric form in which it exists within the cochlea, has the appropriate mechanical properties to serve as a portion of the gating spring. Further, at the physiological Ca2+ concentration within the endolymph ranges from approximately 20 μM at the base to 40 μM at the apex20. Furthermore, the local depletion of Ca2+ might be significant near an open transduction channel: at a distance of 7 nm, the concentration of Ca2+ could fall to half its maximal value. The length of a folded EC domain30 is about 4.5 nm, so EC10, EC11, and the PICA domain could experience a significantly lower Ca2+ level: the behavior of PCDH15 in the distal domains might well lie between the results for physiological levels of Ca2+ and those in the ion’s absence. In particular, for a Ca2+ concentration of 0–20 μM Ca2+, PCDH15 could extend around 20–100 nm. The most extreme tip link extensions observed experimentally37 are around 120 nm, but because mechanotransduction is very sensitive—even hair bundle deflections of 1 nm can produce a response—it is likely that the tip link ordinarily extends much less.The Ca2+, and unfolds to a still more striking extent in the absence of Ca2+. Because the end-to-end distance at low force is far greater for the mutant dimer than for the native dimer, V507D is apparently misfolded at the baseline of 1 pN force. When the applied force is low, such as during the resting period between force-ramp cycles, the two partially unfolded strands of the dimer might tangle with each other, resulting in a misfolded protein that extends easily when force is reapplied. This behavior might occur because EC5 is more prone to unfolding due to the location of the mutation, which could then predispose the neighboring EC domains to unfold by disrupting the stability of the interdomain linkers. Furthermore, the refolding ability of V507D is significantly lower than that of the native protein at physiological and no Ca2+ concentrations for which the comparison is possible. If V507D is unable to refold on a timescale appropriate for normal hearing, on the order of microseconds to milliseconds, a more compliant PCDH15 dimer might underlie a softer overall tip link (2+ through transduction channels, a hair bundle undergoes remodeling, resulting in shorter stereocilia with abnormal tip shapes38. Moreover, if transduction is abolished in inner hair cells, their innervation changes: they become re-innervated by inhibitory efferent neurons, which normally contact inner hair cells only before the onset of hearing39. If V507D cannot convey the appropriate tension to transduction channels and thus prevents their opening, changes in stereociliary structure and synaptic connectivity could underlie deafness.The V507D mutation yields significant unfolding even at a saturating concentration of Catip link . Insuffi2+ in the vestibular labyrinth allows mutated PCDH15 to function adequately, whereas the lower concentration of Ca2+ in the cochlea precludes this. It is also plausible that mutated protein can function adequately at the lower frequencies, less than 20 Hz, characteristic of the vestibular system40, but cannot perform well at the higher frequencies, up to 20 kHz, detected by the cochlea2. PCDH15 V507D might be able to refold on the timescale of low-frequency stimulation in the vestibular system, but not at the higher frequencies experienced in the cochlea. As a result, it is likely that the mutated PCDH15 cannot transmit the appropriate tension to the mechanotransduction channel, resulting in deafness in people carrying this mutation. A similar mechanism might underlie the hearing loss associated with many other mutations16 in PCDH15 and CDH23.While the V507D point mutation disrupts the mechanics of PCDH15, individuals with this mutation retain normal equilibrium and vision, despite the expression of PCDH15 in the vestibule and eye. It is possible that the higher concentration of Ca2+-binding sites are disrupted: because the Ca2+ ions are coordinated by residues both in the linkers and at the edges of the neighboring domains, unfolding of a neighboring domain could disrupt one or more binding sites and liberate Ca2+. The loss of the bound Ca2+ would then predispose the region to further unfolding. Although reducing the force on the protein might allow refolding to occur, proper refolding might become impossible after excessive unfolding.Because PCDH15 consists of repeating structural motifs, it is impossible to ascertain the specific structural origin of any unfolding event. It seems likely that, once a particular part of the molecule has unfolded, the neighboring regions become more vulnerable to unfolding, especially when Ca2+. In the case of a hearing-loss mutation, PCDH15 unfolds much more frequently, is softer than the wild-type protein, and has impaired refolding ability, three features that would likely result in inappropriate tension conveyed to the transduction channels in vivo. The findings concerning the V507D hearing-loss mutation underscore how the tension conveyed to transduction channels is critical for normal hearing.These results confirm that PCDH15 has the appropriate stiffness to form a component of the gating spring and that its physical properties can be modulated by CaSupplement 1


Evidence indicates that intracellular Na+ concentration ([Na+]i) is elevated in breast tumors, and that aberrant Ca2+ signaling regulates numerous key cancer hallmark processes. The present study therefore aimed to determine the effects of Na+ depletion on intracellular Ca2+ handling in metastatic breast cancer cell lines. The relationship between Na+ and Ca2+ was probed using fura‐2 and SBFI fluorescence imaging and replacement of extracellular Na+ with equimolar N‐methyl‐D‐glucamine (0Na+/NMDG) or choline chloride (0Na+/ChoCl). In triple‐negative MDA‐MB‐231 and MDA‐MB‐468 cells and Her2+ SKBR3 cells, but not ER+ MCF‐7 cells, 0Na+/NMDG and 0Na+/ChoCl resulted in a slow, sustained depletion in [Na+]i that was accompanied by a rapid and sustained increase in intracellular Ca2+ concentration ([Ca2+]i). Application of La3+ in nominal Ca2+‐free conditions had no effect on this response, ruling out reverse‐mode NCX activity and Ca2+ entry channels. Moreover, the Na+‐linked [Ca2+]i increase was independent of membrane potential hyperpolarization (NS‐1619), but was inhibited by pharmacological blockade of IP3 receptors (2‐APB), phospholipase C  or following depletion of endoplasmic reticulum Ca2+ stores (cyclopiazonic acid). Thus, Na+ is linked to PLC/IP3‐mediated activation of endoplasmic reticulum Ca2+ release in metastatic breast cancer cells and this may have an important role in breast tumors where [Na+]i is perturbed.Intracellular Ca + concentration is elevated in breast cancer cells, and aberrant Ca2+ signaling regulates numerous key cancer hallmark processes. Here, we found that Na+ depletion in metastatic breast cancer cell lines led to a rapid and sustained increase in intracellular Ca2+ concentration via PLC/IP3‐mediated activation of endoplasmic reticulum Ca2+ release. Our findings suggest that Na+‐dependent alteration of Ca2+ handling may have an important role in breast tumors where Na+ is perturbed.Intracellular Na These channels harness the electrochemical gradient of Na+ and Ca2+ across cell membranes to achieve their function, with [Na+]i and [Ca2+]i typically tightly regulated by various ion transporters  and the mitochondrial Na+/Ca2+/Li+ exchanger  drive metastasis  are linked with breast cancer progression and poor prognosis  or choline chloride (0Na+/ChoCl). These replacement cations maintain the osmotic balance across the cell membrane, yet are not transported via Na+ channels or transporters. Breast cancer cells can be subdivided into three main categories based on expression of the estrogen receptor (ER), progesterone receptor and human epidermal growth factor 2 (Her2). In triple‐negative MDA‐MB‐231 and MDA‐MB‐468 cells  and Her2+ SKBR3 cells, removal of Na+ from the extracellular space led to a slow, sustained depletion in [Na+]i that was accompanied by a rapid and sustained increase in intracellular [Ca2+]i. Interestingly, this response was absent in ER+ MCF‐7 cells. The observed [Ca2+]i transient was not due to Ca2+ entry from the extracellular space, ruling out reverse‐mode NCX activity. Moreover, it was independent of membrane potential (Vm) and was inhibited by depletion of the endoplasmic reticulum Ca2+ store or by pharmacological blockade of inositol  trisphosphate (IP3) receptors (IP3Rs) or phospholipase C (PLC). These data reveal a previously unreported Na+‐linked activation of endoplasmic reticulum Ca2+ release in metastatic breast cancer cells, which may have an important role in breast tumors where [Na+]i is elevated or perturbed. Moreover, the dramatic Ca2+ release observed upon Na+ depletion serves as a cautionary note for those utilizing similar Na+ replacement methods to study the role of Na+ transport in cellular processes.The present study aimed to determine the effects of Na22.12 (95:5%) in Dulbecco's modified Eagle's medium  supplemented with 4 mM L‐glutamine and 5% fetal bovine serum, as described previously  with 0.08% Pluronic F‐127 at room temperature . Cells were then rinsed once with HEPES‐PSS and incubated in dye‐free HEPES‐PSS to allow uncleaved dye to re‐equilibrate . For all experiments except those shown in Supplementary Figure +‐free HEPES PSS was prepared as for HEPES‐PSS above but with NaCl replaced with equimolar N‐methyl‐D‐glucamine (0Na+/NMDG) or choline chloride (0Na+/ChoCl). Nominal Ca2+‐free HEPES PSS was prepared as for HEPES‐PSS above, omitting CaCl2.Cells were seeded onto glass coverslips and allowed to adhere overnight. Cells were loaded with either fura‐2 AM (4 μM) or SBFI AM (4 μM) in HEPES‐buffered physiological saline solution , and ionomycin were prepared in DMSO. The final DMSO concentration in working solutions was 0.1%. Stocks of La2.42+]i in response to treatment were measured as either the maximum fura‐2 ratio change (ΔRmax) or the area under the curve (AUC). The fura‐2 fluorescence ratios for each trace were first normalized to the ratio at time 0 (R/R0) to control for variability in starting ratio between cells. To obtain a pretreatment baseline, the average ratio across the six timepoints  immediately prior to treatment application was determined. ΔRmax was defined as the maximum increase from this baseline during the treatment period. The AUC was defined as the area under the curve during the entire treatment period  in fura‐2 ratio unit seconds (R.s). Where no [Ca2+]i response was elicited in response to treatment, cells were stimulated with ionomycin (3 μM) to ensure a change in [Ca2+]i could be measured. Cells in which no fura‐2 fluorescence change was observed were excluded from analysis.Changes in i  NCX activity  and perfused with sequential pulses of HEPES‐PSS where Na+ had been replaced with either equimolar N‐methyl D glucamine (0Na+/NMDG) or equimolar choline chloride (0Na+/ChoCl) to remove extracellular Na+ while maintaining osmotic balance. Fura‐2 fluorescence imaging revealed that, using either maneuver, removal of extracellular Na+ resulted in a steep increase in [Ca2+]i that returned to baseline upon reperfusion with standard Na+‐containing HEPES‐PSS  or Ca2+ influx from the extracellular space. To determine whether Ca2+ influx from the extracellular space contributed to the [Ca2+]i responses observed, MDA‐MB‐231 cells were perfused with 0Na+/NMDG in nominal Ca2+‐free conditions with La3+ (1 mM); La3+ in the millimolar range is a known inhibitor of both Ca2+ entry from and Ca2+ efflux to the extracellular space  activator NS‐1619 at a concentration (10 μM) that hyperpolarizes Vm in MDA‐MB‐231 cells to a similar degree to 0Na+/NMDG  inhibitor cyclopiazonic acid  in nominal Ca2+‐free conditions (to prevent store‐operated Ca2+ entry), resulting in a transient leak of Ca2+ from the endoplasmic reticulum that was subsequently cleared from the cell . These results indicate that the 0Na+/NMDG‐induced [Ca2+]i transient is likely due to release of Ca2+ from the intracellular endoplasmic reticulum Ca2+ stores.Having ruled out Cal Figure , presuma3.52+ release in the changes in [Ca2+]i observed upon application of 0Na+/NMDG, we next sought to determine the key molecular players responsible. Ca2+ is classically released from the endoplasmic reticulum via inositol  triphosphate receptors (IP3Rs) and ryanodine receptors  with either the IP3R inhibitor 2‐APB (50 μM) or the RyR inhibitor dantrolene (10 μM). Compared to control cells , whereas dantrolene , which then cleaves membrane‐bound phosphatidylinositol 4,5‐bisphosphate (PIP2) to form the second messengers IP3 and diacylglycerol , indicating that PLC activation, potentially via GPCRs, is critical to this Na+‐controlled ER Ca2+ release mechanism.IP4+ depletion and a subsequent dramatic rise in [Ca2+]i that is mediated by a PLC‐ and IP3R‐dependent mechanism. These results provide further evidence of an interplay between Ca2+ and Na+ signaling in breast cancer cells that may have important implications for understanding cancer cell physiology. Through pharmacological maneuvers we rule out any role for reverse‐mode NCX activity, Vm and Ca2+ entry in this response. We also found that CPA abolished the [Ca2+]i transients elicited by 0Na+/NMDG application; CPA is a well characterized SERCA blocker, providing further indirect evidence that this phenomenon was mediated by an IP3R‐mediated endoplasmic reticulum Ca2+ release mechanism. Furthermore, while [Ca2+]i transients were observed upon Na+ depletion in MDA‐MB‐231, MDA‐MB‐468, and SKBR3 cells, no such response was observed in MCF‐7 cells. This finding indicates that the Na+‐dependent Ca2+ increase is not a universal feature across all breast cancer cell lines. Further work is required to determine the underlying reason for the difference in Na+ and Ca2+ signaling between different cancer cell lines.The present study is the first to describe a relationship in metastatic breast cancer cells between Na+ regulates a PLC/IP3‐dependent Ca2+ release in metastatic breast cancer cells; however, the molecular identity of the Na+ “sensor” that initiates this event remains unknown. Interestingly, Na+ has been described as an endogenous regulator of Class A GPCRs  should be taken into careful consideration and controlled for when using 0Na+/NMDG, 0Na+/ChoCl or similar maneuvers to probe Na+ conductance mechanisms.It is worthy to note that, in the present study, the PLC inhibitor U73122 had a more potent inhibitory effect on the [Ca2+‐free extracellular buffer abolished the [Ca2+]i transients elicited by 0Na+/NMDG application, suggesting that EGTA exerts an inhibitory effect on the PLC and IP3‐mediated Ca2+ release upon Na+ depletion. It has previously been shown that EGTA can inhibit mobilization of IP3‐sensitive Ca2+ stores  and/or via fluid‐phase endocytosis and subsequent release into the cytosol , IP3Rs are upregulated in breast cancer (Foulon et al., 3R signaling regulates breast cancer cell proliferation (Szatkowski et al., 3Rs in breast cancer cells led to dysregulated bioenergetics, and autophagy, cell cycle arrest, and cell death (Mound et al., + and Ca2+ handling and their inextricable link via common channels and transporters hint at a hitherto relatively underexplored avenue in the context of cancer. The present study provides further evidence of such a link between Ca2+ and Na+ signaling that may have important implications for regulating cancer cell behavior and future therapy.Beyond the present study, very little is known about the link between CaThe project was designed by WJB and ADJ; experiments were carried out by ADJ, NS, KU, IJ, and RS with the assistance of GJOE; data analysis was performed by ADJ, NS, KU, IJ, and RS; the manuscript was prepared by ADJ and WJB; ADJ, WJB, GJOE, and SC contributed to study design, interpretation of the data, and critical revision of the paper for important intellectual content.The authors declare that they have no competing interests.No animals or human subjects were used in this study.Supplementary Figure S1.Click here for additional data file.Supplementary Figure S2.Click here for additional data file.


Dear Editor,NPM1mut). However, their worldwide availability is limited and they provide limited insight into disease heterogeneity. Hence, surrogate markers of NPM1mut are used for fast diagnostic screening of the disease [NPM1mut) [Molecular techniques are the gold standard method for the diagnosis of AML with mutated nucleophosmin gene NPMmut. Howe disease , cup-lik disease , normal  disease . NeverthNPM1mut) , frequenNPM1mut leukemia cells present with heterogeneous cytomorphology and immunophenotypic patterns of lineage commitment and antigen expression, including phenotypes associated with FLT3-internal tandem duplication (ITD) and a poor outcome [NPM1mut cells has been linked with underlying TET2 and IDH1/2 mutations, while the presence of monocytic lineage-committed and immature NPM1mut cells has been related to DNMT3A mutations; indeed, these three patient subgroups show increasingly worse outcomes [NPM1mutFLT3-ITD− patients displaying immature immunophenotypes show strong similarities with NPM1mutFLT3-ITD+ cases regarding leukemia cell transcriptomic profiles, response to therapy and poorer outcomes [NPM1mut leukemia cell heterogeneity might contribute to guiding treatment decisions in these patients. Despite the above associations, specific immunophenotypic patterns of AML-NPM1mut remain to be fully defined.AML- outcome . In paraoutcomes . Strikinoutcomes . Thus, bNPM1mut, 144 AML-NPM1wt and 32 APL patients, based on the EuroFlow 8-color acute leukemia orientation tube (ALOT) and the AML/MDS antibody panel  immature leukemia cells showing stem cell-like features  neutrophil lineage-committed CD117+/het HLA-DR− (45%), and/or; (3) monocytic-lineage AML cells expressing CD64+/hi HLA-DR+ and variable CD117 levels (54% of cases) , and; (2) mixed expansions of >1 leukemia cell population  , followed by monocytic-lineage (19%) and immature leukemia cells (13%). Conversely, mixed leukemia cell expansions included mixed (1) immature and monocytic (23%), (2) monocytic and neutrophil (7%), (3) immature and neutrophil (5%) and (4) immature plus neutrophil- and monocytic-lineage AML cells (5% of cases).Our data confirm that AML- and/or;  monocytiNPM1mut vs. NPM1wt cells, associated with a lower frequency and size of immature leukemia cell expansions (p < 0.001), while depicting a higher prevalence of more differentiated AML cells committed to the neutrophil (p < 0.001) and/or the monocytic lineage (p = 0.02) (Supplementary Table NPM1mut [The distribution of leukemia cell subsets was consistent with a lower maturation arrest of AML-e p = 0.0 (Supplem NPM1mut .NPM1mut may originate from CD34+ hematopoietic progenitor cells (HPC) [NPM1wt CD34+ cells). These cells may expand in BM due to consistent expression of HOX genes [lo expression is not specific to AML-NPM1mut, and it has been found to be independent of NPM1 dislocation [lo expression among NPM1wt immature, neutrophil and monocytic lineage-committed AML cells, and thereby, this phenotype is of limited specificity for AML-NPM1mut.Despite AML-ls (HPC) , most frOX genes , which hOX genes . Howeverlocation . In linelo expression, other immunophenotypic features of AML-NPM1mut cells supported a less pronounced maturation blockade vs. other AML patients. Hence, AML-NPM1mut immature cells retained a higher capability for neutrophil lineage maturation with higher expression of CyMPO (p = 0.04), CD15 and CD33 (p < 0.001), associated with downregulation of the early monocytic markers CD64 (p = 0.05) and HLA-DR (p = 0.004), in line with the higher frequency of neutrophil (vs. monocytic) lineage-commitment observed for AML-NPM1mut cells. Furthermore, AML-NPM1mut cases more frequently showed immature AML cells with aberrant CD7 positivity (60% vs. 32% cases), but they rarely expressed CD56 (1% vs. 15%) and NuTdT  (p < 0.001)  more immature features, including downregulation of the neutrophil lineage markers CD15, CD71, CD13 and CD64 (p ≤ 0.05), associated with higher levels of the immature antigens CD123 and CD105 (p ≤ 0.01). In contrast to NPM1mut immature leukemia cells, NPM1mut neutrophil-lineage cells barely expressed CD7 but more frequently showed aberrant positivity for CD56  and to a lesser extent also for CD9 and CD4 (p ≤ 0.02). Furthermore, compared with neutrophil-lineage APL cells, AML-NPM1mut neutrophil-lineage cells downregulated CD34, CD13, CD64 and CD71, while they upregulated CD105 (p ≤ 0.001), and aberrant CD56 expression, but showed lower rates of CD203c and CD7 (p ≤ 0.02)  decreased expression of immature markers , while upregulated the monocytic-associated antigens CD4 (p = 0.04), CD11b (p = 0.03), CD15, CD36, and CD300e in addition to CD123 (p ≤ 0.006). However, these more markedly mature monocytic features coexisted with asynchronous downregulation of other monocytic-associated markers  and a higher frequency of AML-NPM1mut cases showing aberrant CD56 (p = 0.03). Altogether, these phenotypes defined three unique asynchronous monocytic maturation profiles present in most AML-NPM1mut cases (90%) vs. a minority of NPM1wt patients : [+CD14−: 74% vs. 3% NPM1wt cases, p < 0.001); and/or, either (2) early expression of CD35 prior CD14 , or; (3) early upregulation of CD14 prior CD35    AML cases with D34+ HPC .FLT3-ITD promoted distinct immunophenotypic profiles in NPM1mut and NPM1wt AML , which might contribute to a fast diagnostic screening of NPM1mut and/or FLT3-ITD in AML, and an improved classification of AML-NPM1mut patients. Further prospective studies are needed to confirm these findings.In summary, the mutational status of Supplementary data


A key feature of advanced motion processing in the primate dorsal stream is the existence of pattern cells—specialized cortical neurons that integrate local motion signals into pattern-invariant representations of global direction. Pattern cells have also been reported in rodent visual cortex, but it is unknown whether the tuning of these neurons results from truly integrative, nonlinear mechanisms or trivially arises from linear receptive fields (RFs) with a peculiar geometry. Here, we show that pattern cells in rat primary (V1) and lateromedial (LM) visual cortex process motion direction in a way that cannot be explained by the linear spatiotemporal structure of their RFs. Instead, their tuning properties are consistent with and well explained by those of units in a state-of-the-art neural network model of the dorsal stream. This suggests that similar cortical processes underlay motion representation in primates and rodents. The latter could thus serve as powerful model systems to unravel the underlying circuit-level mechanisms. Rat visual neurons encode global motion direction of composite visual patterns via nonlinear integration mechanisms. Perceiving the velocity  of visual objects is critical to interact effectively with the environment. From a computational point of view, primary visual cortex can be thought as a bank of local moving-edge detectors. Upstream sensory areas face the nontrivial challenge of extracting object motion direction from the V1 representation. The output of a single localized edge detector is intrinsically ambiguous, since it reflects the projection of the global velocity vector onto the direction that is orthogonal to the orientation detected by the unit. Any information regarding the component of object motion that is parallel to such orientation is lost. Thus, when considered in isolation, the output of each of these edge detectors is compatible with infinite combinations of global directions and speeds and is therefore insufficient to fully specify the velocity of the underlying object. Only by combining  multiple local direction signals of this kind, global object direction and speed can be fully determined. This ambiguity is known in the neuroscientific literature as the “aperture problem”   as pattern cells cannot be overlooked. The most direct way to test for this possibility is to reconstruct the linear receptive fields (RFs) of putative pattern cells and try to predict their plaid and grating responses on that basis. If the geometry of their RFs is the cause of their pattern-like tuning, the responses predicted by linear RFs should still be pattern-like. Conversely, if nonlinear, truly integrative mechanisms are at work, linear RFs should fail to produce pattern-like responses.Our study was designed to (i) address this question about the nature of rodent pattern and component cells, (ii) assess their spatial tuning properties, and (iii) measure their abundance across rat V1 and extrastriate visual areas LM and RL. Finally, the entire data processing pipeline applied to the neuronal data was used to characterize the RF structure and tuning properties of the units of a state-of-the-art neural network model of the monkey dorsal stream  gratings and plaids (with a 120° cross-angle), drifting along 12 equi-spaced directions (from 0° to 330°) and presented at two different spatial and temporal frequencies (SFs = 0.02 and 0.04 cycles per degree (cpd); TFs = 2 and 6 Hz), and (ii) spatiotemporally correlated noise movies . Grating and plaid responses were used to compute direction tuning curves and classify the recorded single units as pattern or component cells, based on the standard approach developed in cat and monkey studies . Direction-selective neurons with Zp significantly higher than 0 and larger than Zc were classified as pattern cells; vice versa, direction-selective units with Zc significantly higher than 0 and larger than Zp were classified as component cells (see Supplementary Text). Neurons that did not meet one of these requirements were labeled as unclassified.We recorded a total of 447, 367, and 412 well-isolated single units from V1, LM, and RL, respectively. Among these neurons, 258 units in V1, 187 units in LM, and 184 units in RL were significantly responsive to gratings or plaids  and were therefore included in the analyses described below. Following previous studies (Zp and Zc) and predicted (Zp′ and Zc′) tuning curves and (ii) the contrast index (CI) used to quantify the sharpness of the STA images  or pattern . All other neurons were considered unclassified . In addition, neurons were labeled according to whether they were sufficiently direction tuned (darker shades), i.e., with a direction selectivity index (DSI) > 0.33—an additional requirement that allows classifying a unit as a component (or pattern) direction-selective cell .The example neurons shown in As shown in P = 0.1870, χ2 test; a tendency toward significance was observed when proportions were computed over the whole pool of responsive units; P = 0.1), we nevertheless found a significant increase of the average value of the pattern index  from V1 (PI = −1.64 ± 0.31) to LM . Together, these observations are suggestive of a shallow, hierarchical buildup of global motion detectors from V1 to LM.Overall, these results confirm that, also in rats, as previously observed in mice, motion-sensitive neurons exist, which can be classified as either component or pattern cells, according to the criteria commonly adopted in the monkey literature, although the incidence of pattern cells is way lower than in primate dorsal stream areas  but not statistically different from each other . The same trend was observed for the pool of unclassified units (gray distribution). Thus, for both component and pattern cells, plaids tended to elicit lower responses than gratings. The implication of this result for understanding the perceptual mechanisms underlying motion integration in rats is examined in Discussion.Next, we checked the extent to which these cell types are affected by cross-orientation suppression, given the relevance of this property to probe alternative perceptual models of motion integration in rats . The lobe count distributions were also statistically different , with a larger proportion of pattern than component cells having RFs with just a single prominent lobe , we restricted the pool of neurons included in this analysis to units that were consistently classified as either component or pattern at both SF = 0.04 and SF = 0.02. This reduced the overall pools of component and pattern cells (merged across the three areas) from, respectively, 98 and 30 . In our case, we empirically observed that the spatial scale of the RFs estimated via STA better matched the gratings with SF = 0.02 cpd (see fig. S1A). We therefore computed predictions for responses to gratings and plaids with such SF. To guarantee the consistency with the analysis shown in Zc/Zp plane (light green and light orange dots) and shows how the pairs of Zc and Zp values obtained for each unit changed when it was computed on the direction tuning curves predicted by the LN model (dark green and dark orange dots). The difference between the two populations was striking. While most component cells remained in the “component” region of the plane and those that changed category became at most unclassified, none of the pattern cells retained its classification and many of them ended up being classified as component. This is better quantified by the bar plot in P = 5.9 × 10−11, χ2 test) different, thus testifying the inability of the linear RFs estimated via STA to capture the patternness of pattern cells.Zc and Zp indexes, as computed based on the observed responses (light shading) and the predicted ones (dark shading). In the case of component cells . By contrast, for pattern cells : from close to zero  to close to 2 . This, together with the trend observed for Zp (see below), explains why many pattern cells switched their status from pattern to component when the responses of the LN model were considered . However, this increase was marginal and the index remained substantially and significantly lower than the large positive values (close to 2) observed for pattern cells . This explains why most component cells retained their classification when Zc and Zp were computed based on their predicted responses  obtained for the observed responses . This explains why none of the pattern cells retained its pattern status when the predicted responses were considered  invariance , which quantifies the extent to which the neuronal response of a unit is modulated at the TF of its preferred grating . This shows a general tendency of the two forms of nonlinearity  to co-occur in rat visual cortical areas. At the same time, one kind of nonlinearity does not necessarily imply the other, as expected for two computations that are critical for two different processing streams (see the Discussion).These examples illustrate how, in our recorded populations, both component and pattern cells could be found that displayed spatial integration properties consistent with either a simple-like or a complex-like behavior. At the population level, pattern cells had a slightly but significantly lower median MI than component cells  but not statistically different from each other , as already observed for rat visual cortical neurons , better approximated by Gabor filters , and better capable to predict the observed Zp and Zc values , and, therefore, to preserve the units’ classification , as compared to the RFs obtained for pattern units. All these trends matched strikingly well those observed for rat component and pattern cells  were sharper . The same applied to the fraction of complex units in the two populations (fig. S2C). The MI, however, changed considerably across the depth of the network (fig. S2D), dropping from being slightly above 3 in the first two layers (consistently with simple-like tuning) to values between 2 and 2.8 in the following layers (consistently with complex-like tuning). This trend was observed across all three populations of component, pattern, and unclassified units, with the fraction of simple cells dropping from 60 to 100% in the first two layers to 0 to 40% in the following ones (fig. S2E). Overall, this result reinforces the intuition that the nonlinear pooling mechanisms underlying the emergence of pattern cells and complex cells are independent processes that can coexist in hierarchical image-processing systems, but one does not necessarily imply the other (see Discussion).We also checked whether the spatial tuning of DorsalNet units displayed any tendency to become more phase-invariant across the layers and whether such tendency was stronger for the population of pattern than component units. As for the case of rat visual cortical neurons , we founThe comparison with DorsalNet carried out in the previous section was at level of emergent properties. That is, we asked whether prominent differences between the tuning properties of the populations of pattern and component DorsalNet units could be found that were consistent with those observed in rat visual cortex—discovering, in most cases, a very good match. We have successfully applied this approach in previous comparisons of processing along the rat ventral stream and deep neural networks for image classification . That is, we modeled the responses of each cell to drifting gratings and plaids as a linear combination of the responses of DN units to these same stimuli (see Supplementary Text). As regressors in this linear modeling scheme, we used either the subpopulation of DN component units or the subpopulation of DN pattern units. This allowed assessing the extent to which the tuning of a cortical neuronal subpopulation  was captured by the tuning of the matching (or unmatching) subpopulation of units in the network.Zc and Zp indexes  extracted from these average curves, as well as the large negative value of the PI nicely captured the component nature of the representation.The rows of the matrix shown in Zc, Zp, and PI indexes obtained from the predicted average curves matched very well the sign and magnitude of those extracted from the measured ones. By contrast, DN pattern units completely failed to capture the tuning of component cells .We found the opposite scenario when we tried to predict the tuning of pattern cells with the activations of DN component and pattern units. Zp and Zc indexes derived from the average curves were small and very similar, yielding a modest PI—inconsistent with that expected for a pattern cell  and Zp (right plots) indexes derived from the DorsalNet predictions were with those obtained from the measured tuning curves for the component (in green) and pattern (in orange) populations . Conversely, for the pattern population, the predicted Zp index became lower than the measured one (dark versus light orange dots), consistently with the failure of this model to capture the tuning for plaid direction , while both indexes changed considerably for the population of component cells (dark versus light green dots). As a result, almost 70% of the pattern cells were correctly predicted as such , it may indicate that different  mechanisms give rise to pattern cells in rodents, as compared to monkeys.The DorsalNet model also allowed assessing the functional properties of the cells that could be classified neither as component nor as pattern. Such unclassified neurons are way more prominent in rat (and mouse) visual cortex than in monkey dorsal stream areas . A possiTo better explore the nature of unclassified cells, we compared the performance of the three different DorsalNet models described in the previous section at capturing their tuning. The model based on the activations of DN unclassified units performed poorly not only at predicting the responses of rat component and pattern cells . This suggests that the unclassified cells that were better modeled by DN component and pattern units were far from being proper component or pattern cells.While this result seems consistent with the continuum scenario described above, when we color-coded the best-fitting model for each rat neuron within the rn units . To bettZc and Zp indexes, resulting in close-to-zero PIs.To further verify this, we selected two subpopulations of unclassified cells for which the model predictions yielded by DN component and pattern units had high confidence (>0.3). We plotted the tuning curves of these pools of quasi-component and quasi-pattern cells using the same layout of In conclusion, our analysis show that rat unclassified cells have a tuning that is not properly accounted by any model we tested—not by DN unclassified regressors, likely because of the heterogeneity of both the cortical and artificial unclassified populations in terms of their selectivity for grating and plaid direction, nor by DN component and pattern regressors, which did marginally better, likely because of their regular tuning curves, but failed to identify unclassified cells with a proper component- or pattern-like behavior see . The impThe first goal of our study was to measure the incidence of pattern and component cells in some visual cortical areas of the rat that, by analogy with previous mouse studies  are in aet al. , as compared to single-unit, cell-tailored recordings.Zc and Zp indexes using, for each cell, the combination of SF and TF that yielded the strongest response at the preferred grating orientation. That is, we did optimize the stimulus parameters, although within our limited range of tested TFs and SFs, before computing the metrics for the classification of the units as either pattern or component. This assured that, for each unit, the Zc and Zp indexes were computed using reliable, visually driven responses, resulting in clearly stimulus-modulated tuning curves, as shown by the examples of Second, we computed the Zc and Zp metrics used to assess such consistency are based on computing Pearson correlation coefficients, which are, by definition, scale invariant. That is, the Zc and Zp metrics are strongly sensitive to the relative position of the peaks along the tuning curves, but not to their magnitude. To our knowledge, the only way by which surround modulation mechanisms may shift the positions of peaks along a tuning curve is when the surround stimulus is appositely designed to only interfere with one of the constituent features of the central stimulus. For instance, Trott and Born , rather than through a circular window, as typical of monkey studies, is the possibility that the neurons’ RFs overlapped the edges of the display. The intersections between these edges and the contours of the gratings could provide unambiguous  pattern direction clues (known as “terminators”), as in the well-known barber-pole illusion, where gratings drifting diagonally are perceived as drifting along the longer edge of a rectangular aperture . Still, The second goal of our study was to characterize some key spatial integration properties of rat pattern and component cells. We started by assessing the extent to which cross-orientation suppression affects component and pattern cells in rat visual cortex. Addressing this question is important for two reasons. First, cross-orientation suppression belongs to a class of nonlinear tuning phenomena that are thought to depend on divisive normalization, a canonical cortical computation where the response of a neuron is divided  by the summed activity of a pool of nearby neurons. Divisive normalization has been called into cause to explain a variety of nonlinear interactions among competing stimuli within the classical and extraclassical RFs of visual neurons in cats and monkeys  simple cells. To our knowledge, the relationship between pattern/component and complex/simple cells has not been systematically explored in the monkey literature. On the basis of computational considerations . This is not surprising, since that network was designed to solve a typical dorsal-processing task and, as such, lacked a specific module devoted to ventral processing. The fact that DorsalNet units nevertheless displayed a tendency to become more complex-like in deep layers suggests that acquiring some degree of translation invariance is useful for motion-related computations. At the same time, the fact that phase invariance increase of virtually the same amount for pattern and component units indicates, once again, that the computations underlying pattern-invariant encoding of motion direction and translation-invariant encoding of orientation are independent.The last and more important goal of our study was to assess whether pattern cells in rodents perform a truly integrative, nonlinear processing of local motion signals to encode global motion direction of complex patterns, as the plaid stimuli. Answering this question is important because, as originally pointed out by  to reconstruct the linear RF of units classified as either component or pattern cells by processing their responses to spatiotemporally correlated noise movies . As expected, for many component cells, we obtained temporal sequences of high-contrast STA images , typicalirically . As a rehe model . Also, phe model . Howeverhe model . This suhe model  are the he model . On the This conclusion has important implications for our understanding of the nature of motion processing in rodent visual cortex. Looking at the primate dorsal stream literature, the most established mechanistic models of pattern-motion selectivity in MT are based on the nonlinear pooling of inputs from narrowly tuned, V1-like component cells, whose preferred motion directions are spread over a wide range of angles  the presence of both component and pattern units, with an increase in abundance of the latter in higher stages of processing, possibly reflecting what we observed between V1 and LM compare ; (ii) thTo further strengthen this conclusion, we also modeled (via linear regression) the tuning of rat component and pattern cells for grating and plaid direction using the activations of component and pattern DN units. DN component units successfully captured the tuning of rat component cells, but not of pattern cells. Vice versa, DN pattern units accounted for the tuning of pattern cells, but not of component cells. This confirms that the functional properties of rat pattern cells can only be captured by models that are capable to encode global motion direction (like the pool of DN pattern units)—models encoding local direction only (like the STA-based LN models or the set of DN component units) are not up to the task. At the same time, our results show that the reverse is also true—global motion encoders do not succeed at modeling the selectivity of local motion encoders, as rat component cells. This may explain why in multiple species , as well as in DorsalNet, populations of pattern and component cells coexist at every processing stage . Maintaining the ability to extract the direction of the elemental, constituent features (the components) of global patterns could be of some functional relevance in the processing of motion information. If the hierarchical buildup of motion integration would ultimately result in a population of pattern cells only, sensitivity to the local direction of the component features would be fully lost.Zc/Zp plane. Rather, the class of pattern cells seems to be the result of dedicated nonlinear integrative mechanisms, possibly not dissimilar from those at work in the monkey dorsal stream.We also tried to predict the tuning of rat unclassified cells, using various subpopulations of DN regressors, but neither the unclassified units nor the component or pattern units yielded good fits . Our modIn summary, our experimental findings, supported by the comparison with the tuning properties of DN units, provide compelling evidence for the existence of truly advanced encoders of global motion direction distributed across rat visual cortical areas V1 and LM. As such, our study sets the stage to exploit the molecular and genetic tools that are available in rodents to dissect the circuit-level mechanisms underlying integration of local motion signals  for sessions targeting V1, 7 mm AP and 5 mm ML for sessions targeting ML, and 5 mm AP and 4.5 mm ML for sessions targeting RL.We performed extracellular neuronal recordings from 29 naïve male Long Evans rats, weighted 300 to 700 g and aged 3 to 12 months. Each rat was anesthetized with an intraperitoneal injection of a solution of 0.3 mg/kg of fentanyl  and 0.3 mg/kg of medetomidin . During the surgery, we monitored the anesthesia level by checking the animal paw reflex and by measuring the oxygenation and heart rate through a pulse oximeter . Temperature was monitored and maintained at 37°C through a heating pad, and a constant flux of oxygen was delivered to the animal throughout the surgery. A constant level of anesthesia was maintained by continuously delivering an intraperitoneal injection of the same aesthetic solution used for the induction, but at a lower concentration (0.1 mg/kg per hour of fentanyl and 0.1 g/kg per hour of medetomidin), by means of a syringe pump . Once deeply anesthetized, the animal was secured to a stereotaxic apparatus  and we performed a craniotomy on the left hemisphere, over the selected target . Once the surgery was completed, the rat was placed over a rotating platform (used to place the RFs of the recorded neurons on the center of the monitor), with the right eye just in front of the center of the screen (distance = 30 cm) and the left eye covered with nontransparent black tape. The right eye was fixed through a metal eye-ring to prevent eye movements during the visual stimulation protocol.2 and an intersite spacing of 25 μm. The insertion of the electrode into the cortex was performed through an oil hydraulic micromanipulator . The insertion depth was different for each area: For V1 and RL, it was ~900 μm with an insertion angle relative to the cortical surface of ~20°; for LM, it was ~1500 μm, with an insertion angle of ~25°. Neuronal signals were recorded with a sampling rate of 25 kHz, and were acquired and preamplified using a system three TDT (Tucker-Davis Technologies) workstation.Extracellular recordings were performed under light anesthesia while rats were passively exposed to visual stimulation. Recordings were carried out using single-shank (or double-shank) 32-channel (or 64-channel) silicon probes (NeuroNexus Technologies) with a site recording area of 775 μmHere, we did not reconstruct the cortical depth and laminar location of the recording sites. However, based on our previous work, where recordings were performed using the same experimental approach, we can speculate that most cells were recorded from layer 5 and, to a lesser extent, from layer 4 (z-scored points relative to baseline activity. The average baseline (spontaneous) firing rate of each well-isolated unit was computed by averaging its spiking activity over every interstimulus interval.Single units were isolated offline using the KlustaKwik-Phy software package  and shown against a black background in 66 different positions, across a grid of 6 rows  and 11 columns . This procedure allowed identifying the visual area each unit was recorded from, by tracking the reversals of the retinotopy that, in rodents, take place at the boundaries between adjacent visual areas, as described in several other studies from our group . Grating stimuli were presented at 60-Hz refresh rate, whereas noise movies were played at 30 Hz.The second visual stimulation protocol included all the stimuli used to characterize neurons as pattern or component cells and to reconstruct their linear RFs, as described in the main text of this paper. The protocol consisted of (i) 20 repetitions  of 1.5-s-long, full-contrast drifting gratings, made of all possible combinations of two SFs (0.02 and 0.04 cpd), two TFs (2 and 6 Hz), and 12 directions ; (ii) 20 repetitions of 1.5-s-long drifting plaids , again spanning all possible combinations of two SFs (0.02 and 0.04 cpd), two TFs (2 and 6 Hz), and 12 directions ; and (iii) 20 different 60-s-long spatially and temporally correlated, as well as contrast-modulated noise movies, which were built as described in  was defined as the trial-averaged firing rate, computed over the entire stimulus presentation window and z-scored with respect to the spontaneous activity, as computed during all interstimulus intervals . Neurons with DSI > 0.33 were categorized as direction selective.We quantified direction selectivity of single units in each area by computing the DSI:f1 of a drifting grating was quantified by an MI adapted from , whose definition, derived from the monkey dorsal stream literature  and (re) classify each unit as component, pattern, or unclassified  of the response of each unit to the plaid as compared to the grating stimuli, we defined a normalized CSI as:Rp and Rc indicate the peak responses to plaids and gratings, respectively . This index takes a value of 1 for a unit responsive to gratings but not to plaids  and, vice versa, a value of −1 for a unit responsive to plaids but not to gratings , whereas it takes a value of 0 for a unit showing the same peak response firing rate for both gratings and plaids. To correctly interpret this index, attention should be paid to the fact that both gratings and plaids were presented at full contrast. This was done to elicit stronger responses and increase the yield of the recordings, as well as to match the contrast of the stimuli used in our previous behavioral study  as well as using MATLAB built-in statistical functions “ttest2,” “signrank,” and “ranksum” (for t tests and Wilcoxon tests). The dataset includes data from 29 animals for a total of 447, 367, and 412 well-isolated single units in areas V1, LM, and RL, respectively. Out of this, 258, 187, and 184 were selected as stimulus responsive and included in the analyses presented in the study. The sample size of different neuronal subpopulations meeting specific response-property criteria that were used for each analysis is reported in the main text.All statistical analyses were computed in MATLAB (MathWorks) 2018a and 2019b using custom software (implementing STA permutation test and