Decreased expression of synaptic genes in the vestibular ganglion of rodents following subchronic ototoxic stress.

The vestibular ganglion contains primary sensory neurons that are postsynaptic to the transducing hair cells (HC) and project to the central nervous system. Understanding the response of these neurons to HC stress or loss is of great interest as their survival and functional competence will determine the functional outcome of any intervention aiming at repair or regeneration of the HCs. We have shown that subchronic exposure to the ototoxicant 3,3'-iminodipropionitrile (IDPN) in rats and mice causes a reversible detachment and synaptic uncoupling between the HCs and the ganglion neurons. Here, we used this paradigm to study the global changes in gene expression in vestibular ganglia using RNA-seq. Comparative gene ontology and pathway analyses of the data from both model species indicated a robust downregulation of terms related to synapses, including presynaptic and postsynaptic functions. Manual analyses of the most significantly downregulated transcripts identified genes with expressions related to neuronal activity, modulators of neuronal excitability, and transcription factors and receptors that promote neurite growth and differentiation. For choice selected genes, the mRNA expression results were replicated by qRT-PCR, validated spatially by RNA-scope, or were demonstrated to be associated with decreased expression of the corresponding protein. We conjectured that decreased synaptic input or trophic support on the ganglion neurons from the HC was triggering these expression changes. To support this hypothesis, we demonstrated decreased expression of BDNF mRNA in the vestibular epithelium after subchronic ototoxicity and also downregulated expression of similarly identified genes (e.g Etv5, Camk1g, Slc17a6, Nptx2, Spp1) after HC ablation with another ototoxic compound, allylnitrile. We conclude that vestibular ganglion neurons respond to decreased input from HCs by decreasing the strength of all their synaptic contacts, both as postsynaptic and presynaptic players.

The vestibular calyceal junction is dismantled following subchronic streptomycin in rats and sensory epithelium stress in humans.

Hair cell (HC) loss by epithelial extrusion has been described to occur in the rodent vestibular system during chronic 3,3'-iminodipropionitrile (IDPN) ototoxicity. This is preceded by dismantlement of the calyceal junction in the contact between type I HC (HCI) and calyx afferent terminals. Here, we evaluated whether these phenomena have wider significance. First, we studied rats receiving seven different doses of streptomycin, ranging from 100 to 800 mg/kg/day, for 3-8 weeks. Streptomycin caused loss of vestibular function associated with partial loss of HCI and decreased expression of contactin-associated protein (CASPR1), denoting calyceal junction dismantlement, in the calyces encasing the surviving HCI. Additional molecular and ultrastructural data supported the conclusion that HC-calyx detachment precede HCI loss by extrusion. Animals allowed to survive after the treatment showed functional recuperation and rebuilding of the calyceal junction. Second, we evaluated human sensory epithelia obtained during therapeutic labyrinthectomies and trans-labyrinthine tumour excisions. Some samples showed abnormal CASPR1 label strongly suggestive of calyceal junction dismantlement. Therefore, reversible dismantlement of the vestibular calyceal junction may be a common response triggered by chronic stress, including ototoxic stress, before HCI loss. This may partly explain clinical observations of reversion in function loss after aminoglycoside exposure.

Assessment of cochlear toxicity in response to chronic 3,3'-iminodipropionitrile in mice reveals early and reversible functional loss that precedes overt histopathology.

The peripheral auditory and vestibular systems rely on sensorineural structures that are vulnerable to ototoxic agents that cause hearing loss and/or equilibrium deficits. Although attention has focused on hair cell loss as the primary pathology underlying ototoxicity, evidence from the peripheral vestibular system indicates that hair cell loss during chronic exposure is preceded by synaptic uncoupling from the neurons and is potentially reversible. To determine if synaptic pathology also occurs in the peripheral auditory system, we examined the extent, time course, and reversibility of functional and morphological alterations in cochleae from mice exposed to 3,3'-iminodipropionitrile (IDPN) in drinking water for 2, 4 or 6 weeks. Functionally, IDPN exposure caused progressive high- to low-frequency hearing loss assessed by measurement of auditory brainstem response wave I absolute thresholds and amplitudes. The extent of hearing loss scaled with the magnitude of vestibular dysfunction assessed behaviorally. Morphologically, IDPN exposure caused progressive loss of outer hair cells (OHCs) and synapses between the inner hair cells (IHCs) and primary auditory neurons. In contrast, IHCs were spared from ototoxic damage. Importantly, hearing loss consistent with cochlear synaptopathy preceded loss of OHCs and synapses and, moreover, recovered if IDPN exposure was stopped before morphological pathology occurred. Our observations suggest that synaptic uncoupling, perhaps as an early phase of cochlear synaptopathy, also occurs in the peripheral auditory system in response to IDPN exposure. These findings identify novel mechanisms that contribute to the earliest stages of hearing loss in response to ototoxic agents and possibly other forms of acquired hearing loss.

Assessing the Performance of RGB-D Sensors for 3D Fruit Crop Canopy Characterization under Different Operating and Lighting Conditions.

The use of 3D sensors combined with appropriate data processing and analysis has provided tools to optimise agricultural management through the application of precision agriculture. The recent development of low-cost RGB-Depth cameras has presented an opportunity to introduce 3D sensors into the agricultural community. However, due to the sensitivity of these sensors to highly illuminated environments, it is necessary to know under which conditions RGB-D sensors are capable of operating. This work presents a methodology to evaluate the performance of RGB-D sensors under different lighting and distance conditions, considering both geometrical and spectral (colour and NIR) features. The methodology was applied to evaluate the performance of the Microsoft Kinect v2 sensor in an apple orchard. The results show that sensor resolution and precision decreased significantly under middle to high ambient illuminance (>2000 lx). However, this effect was minimised when measurements were conducted closer to the target. In contrast, illuminance levels below 50 lx affected the quality of colour data and may require the use of artificial lighting. The methodology was useful for characterizing sensor performance throughout the full range of ambient conditions in commercial orchards. Although Kinect v2 was originally developed for indoor conditions, it performed well under a range of outdoor conditions.

Calyx junction dismantlement and synaptic uncoupling precede hair cell extrusion in the vestibular sensory epithelium during sub-chronic 3,3'-iminodipropionitrile ototoxicity in the mouse.

The cellular and molecular events that precede hair cell (HC) loss in the vestibular epithelium during chronic ototoxic exposure have not been widely studied. To select a study model, we compared the effects of sub-chronic exposure to different concentrations of 3,3'-iminodipropionitrile (IDPN) in the drinking water of two strains of mice and of both sexes. In subsequent experiments, male 129S1/SvImJ mice were exposed to 30 mM IDPN for 5 or 8 weeks; animals were euthanized at the end of the exposure or after a washout period of 13 weeks. In behavioral tests, IDPN mice showed progressive vestibular dysfunction followed by recovery during washout. In severely affected animals, light and electron microscopy observations of the vestibular epithelia revealed HC extrusion towards the endolymphatic cavity. Comparison of functional and ultrastructural data indicated that animals with fully reversible dysfunction did not have significant HC loss or stereociliary damage, but reversible dismantlement of the calyceal junctions that characterize the contact between type I HCs (HCI) and their calyx afferents. Immunofluorescent analysis revealed the loss of calyx junction proteins, Caspr1 and Tenascin-C, during exposure and their recovery during washout. Synaptic uncoupling was also recorded, with loss of pre-synaptic Ribeye and post-synaptic GluA2 puncta, and differential reversibility among the three different kinds of synaptic contacts existing in the epithelium. qRT-PCR analyses demonstrated that some of these changes are at least in part explained by gene expression modifications. We concluded that calyx junction dismantlement and synaptic uncoupling are early events in the mouse vestibular sensory epithelium during sub-chronic IDPN ototoxicity.

Special Issue on "Terrestrial Laser Scanning": Editors' Notes.

In this editorial, we provide an overview of the content of the special issue on "Terrestrial Laser Scanning". The aim of this Special Issue is to bring together innovative developments and applications of terrestrial laser scanning (TLS), understood in a broad sense. Thus, although most contributions mainly involve the use of laser-based systems, other alternative technologies that also allow for obtaining 3D point clouds for the measurement and the 3D characterization of terrestrial targets, such as photogrammetry, are also considered. The 15 published contributions are mainly focused on the applications of TLS to the following three topics: TLS performance and point cloud processing, applications to civil engineering, and applications to plant characterization.

Testing the Suitability of a Terrestrial 2D LiDAR Scanner for Canopy Characterization of Greenhouse Tomato Crops.

Canopy characterization is essential for pesticide dosage adjustment according to vegetation volume and density. It is especially important for fresh exportable vegetables like greenhouse tomatoes. These plants are thin and tall and are planted in pairs, which makes their characterization with electronic methods difficult. Therefore, the accuracy of the terrestrial 2D LiDAR sensor is evaluated for determining canopy parameters related to volume and density and established useful correlations between manual and electronic parameters for leaf area estimation. Experiments were performed in three commercial tomato greenhouses with a paired plantation system. In the electronic characterization, a LiDAR sensor scanned the plant pairs from both sides. The canopy height, canopy width, canopy volume, and leaf area were obtained. From these, other important parameters were calculated, like the tree row volume, leaf wall area, leaf area index, and leaf area density. Manual measurements were found to overestimate the parameters compared with the LiDAR sensor. The canopy volume estimated with the scanner was found to be reliable for estimating the canopy height, volume, and density. Moreover, the LiDAR scanner could assess the high variability in canopy density along rows and hence is an important tool for generating canopy maps.

Testing accuracy of long-range ultrasonic sensors for olive tree canopy measurements.

Ultrasonic sensors are often used to adjust spray volume by allowing the calculation of the crown volume of tree crops. The special conditions of the olive tree require the use of long-range sensors, which are less accurate and faster than the most commonly used sensors. The main objectives of the study were to determine the suitability of the sensor in terms of sound cone determination, angle errors, crosstalk errors and field measurements. Different laboratory tests were performed to check the suitability of a commercial long-range ultrasonic sensor, as were the experimental determination of the sound cone diameter at several distances for several target materials, the determination of the influence of the angle of incidence of the sound wave on the target and distance on the accuracy of measurements for several materials and the determination of the importance of the errors due to interference between sensors for different sensor spacings and distances for two different materials. Furthermore, sensor accuracy was tested under real field conditions. The results show that the studied sensor is appropriate for olive trees because the sound cone is narrower for an olive tree than for the other studied materials, the olive tree canopy does not have a large influence on the sensor accuracy with respect to distance and angle, the interference errors are insignificant for high sensor spacings and the sensor's field distance measurements were deemed sufficiently accurate.

Towards an optimized method of olive tree crown volume measurement.

Accurate crown characterization of large isolated olive trees is vital for adjusting spray doses in three-dimensional crop agriculture. Among the many methodologies available, laser sensors have proved to be the most reliable and accurate. However, their operation is time consuming and requires specialist knowledge and so a simpler crown characterization method is required. To this end, three methods were evaluated and compared with LiDAR measurements to determine their accuracy: Vertical Crown Projected Area method (VCPA), Ellipsoid Volume method (VE) and Tree Silhouette Volume method (VTS). Trials were performed in three different kinds of olive tree plantations: intensive, adapted one-trunked traditional and traditional. In total, 55 trees were characterized. Results show that all three methods are appropriate to estimate the crown volume, reaching high coefficients of determination: R2 = 0.783, 0.843 and 0.824 for VCPA, VE and VTS, respectively. However, discrepancies arise when evaluating tree plantations separately, especially for traditional trees. Here, correlations between LiDAR volume and other parameters showed that the Mean Vector calculated for VCPA method showed the highest correlation for traditional trees, thus its use in traditional plantations is highly recommended.

Advanced technologies for the improvement of spray application techniques in spanish viticulture: an overview.

Spraying techniques have been undergoing continuous evolution in recent decades. This paper presents part of the research work carried out in Spain in the field of sensors for characterizing vineyard canopies and monitoring spray drift in order to improve vineyard spraying and make it more sustainable. Some methods and geostatistical procedures for mapping vineyard parameters are proposed, and the development of a variable rate sprayer is described. All these technologies are interesting in terms of adjusting the amount of pesticides applied to the target canopy.

Maturation of type I and type II rat vestibular hair cells in vivo and in vitro.

Vestibular sensory epithelia contain type I and type II sensory hair cells (HCI and HCII). Recent studies have revealed molecular markers for the identification of these cells, but the precise composition of each vestibular epithelium (saccule, utricle, lateral crista, anterior crista, posterior crista) and their postnatal maturation have not been described in detail. Moreover, in vitro methods to study this maturation are not well developed. We obtained total HCI and HCII counts in adult rats and studied the maturation of the epithelia from birth (P0) to postnatal day 28 (P28). Adult vestibular epithelia hair cells were found to comprise ∼65% HCI expressing osteopontin and PMCA2, ∼30% HCII expressing calretinin, and ∼4% HCII expressing SOX2 but neither osteopontin nor calretinin. At birth, immature HCs express both osteopontin and calretinin. P28 epithelia showed an almost adult-like composition but still contained 1.3% of immature HCs. In addition, we obtained free-floating 3D cultures of the epithelia at P1, which formed a fluid-filled cyst, and studied their survival and maturation in vitro up to day 28 (28 DIV). These cultures showed good HC resiliency and maturation. Using an enriched medium for the initial 4 days, a HCI/calretinin+-HCII ratio close to the in vivo ratio was obtained. These cultures are suitable to study HC maturation and mature HCs in pharmacological, toxicological and molecular research.

Use of a terrestrial LIDAR sensor for drift detection in vineyard spraying.

The use of a scanning Light Detection and Ranging (LIDAR) system to characterize drift during pesticide application is described. The LIDAR system is compared with an ad hoc test bench used to quantify the amount of spray liquid moving beyond the canopy. Two sprayers were used during the field test; a conventional mist blower at two air flow rates (27,507 and 34,959 m3·h(-1)) equipped with two different nozzle types (conventional and air injection) and a multi row sprayer with individually oriented air outlets. A simple model based on a linear function was used to predict spray deposit using LIDAR measurements and to compare with the deposits measured over the test bench. Results showed differences in the effectiveness of the LIDAR sensor depending on the sprayed droplet size (nozzle type) and air intensity. For conventional mist blower and low air flow rate; the sensor detects a greater number of drift drops obtaining a better correlation (r = 0.91; p < 0.01) than for the case of coarse droplets or high air flow rate. In the case of the multi row sprayer; drift deposition in the test bench was very poor. In general; the use of the LIDAR sensor presents an interesting and easy technique to establish the potential drift of a specific spray situation as an adequate alternative for the evaluation of drift potential.

Multisensory gaze stabilization in response to subchronic alteration of vestibular type I hair cells.

The functional complementarity of the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) allows for optimal combined gaze stabilization responses (CGR) in light. While sensory substitution has been reported following complete vestibular loss, the capacity of the central vestibular system to compensate for partial peripheral vestibular loss remains to be determined. Here, we first demonstrate the efficacy of a 6-week subchronic ototoxic protocol in inducing transient and partial vestibular loss which equally affects the canal- and otolith-dependent VORs. Immunostaining of hair cells in the vestibular sensory epithelia revealed that organ-specific alteration of type I, but not type II, hair cells correlates with functional impairments. The decrease in VOR performance is paralleled with an increase in the gain of the OKR occurring in a specific range of frequencies where VOR normally dominates gaze stabilization, compatible with a sensory substitution process. Comparison of unimodal OKR or VOR versus bimodal CGR revealed that visuo-vestibular interactions remain reduced despite a significant recovery in the VOR. Modeling and sweep-based analysis revealed that the differential capacity to optimally combine OKR and VOR correlates with the reproducibility of the VOR responses. Overall, these results shed light on the multisensory reweighting occurring in pathologies with fluctuating peripheral vestibular malfunction.

Hair cell toxicology: With the help of a little fish.

Hearing or balance loss are disabling conditions that have a serious impact in those suffering them, especially when they appear in children. Their ultimate cause is frequently the loss of function of mechanosensory hair cells in the inner ear. Hair cells can be damaged by environmental insults, like noise or chemical agents, known as ototoxins. Two of the most common ototoxins are life-saving medications: cisplatin against solid tumors, and aminoglycoside antibiotics to treat infections. However, due to their localization inside the temporal bone, hair cells are difficult to study in mammals. As an alternative animal model, zebrafish larvae have hair cells similar to those in mammals, some of which are located in a fish specific organ on the surface of the skin, the lateral line. This makes them easy to observe in vivo and readily accessible for ototoxins or otoprotective substances. These features have made possible advances in the study of the mechanisms mediating ototoxicity or identifying new potential ototoxins. Most importantly, the small size of the zebrafish larvae has allowed screening thousands of molecules searching for otoprotective agents in a scale that would be highly impractical in rodent models. The positive hits found can then start the long road to reach clinical settings to prevent hearing or balance loss.

Ultrasonic and LIDAR sensors for electronic canopy characterization in vineyards: advances to improve pesticide application methods.

Canopy characterization is a key factor to improve pesticide application methods in tree crops and vineyards. Development of quick, easy and efficient methods to determine the fundamental parameters used to characterize canopy structure is thus an important need. In this research the use of ultrasonic and LIDAR sensors have been compared with the traditional manual and destructive canopy measurement procedure. For both methods the values of key parameters such as crop height, crop width, crop volume or leaf area have been compared. Obtained results indicate that an ultrasonic sensor is an appropriate tool to determine the average canopy characteristics, while a LIDAR sensor provides more accuracy and detailed information about the canopy. Good correlations have been obtained between crop volume (C(VU)) values measured with ultrasonic sensors and leaf area index, LAI (R(2) = 0.51). A good correlation has also been obtained between the canopy volume measured with ultrasonic and LIDAR sensors (R(2) = 0.52). Laser measurements of crop height (C(HL)) allow one to accurately predict the canopy volume. The proposed new technologies seems very appropriate as complementary tools to improve the efficiency of pesticide applications, although further improvements are still needed.

Georeferenced LiDAR 3D vine plantation map generation.

The use of electronic devices for canopy characterization has recently been widely discussed. Among such devices, LiDAR sensors appear to be the most accurate and precise. Information obtained with LiDAR sensors during reading while driving a tractor along a crop row can be managed and transformed into canopy density maps by evaluating the frequency of LiDAR returns. This paper describes a proposed methodology to obtain a georeferenced canopy map by combining the information obtained with LiDAR with that generated using a GPS receiver installed on top of a tractor. Data regarding the velocity of LiDAR measurements and UTM coordinates of each measured point on the canopy were obtained by applying the proposed transformation process. The process allows overlap of the canopy density map generated with the image of the intended measured area using Google Earth(®), providing accurate information about the canopy distribution and/or location of damage along the rows. This methodology was applied and tested on different vine varieties and crop stages in two important vine production areas in Spain. The results indicate that the georeferenced information obtained with LiDAR sensors appears to be an interesting tool with the potential to improve crop management processes.

Performance of an ultrasonic ranging sensor in apple tree canopies.

Electronic canopy characterization is an important issue in tree crop management. Ultrasonic and optical sensors are the most used for this purpose. The objective of this work was to assess the performance of an ultrasonic sensor under laboratory and field conditions in order to provide reliable estimations of distance measurements to apple tree canopies. To this purpose, a methodology has been designed to analyze sensor performance in relation to foliage ranging and to interferences with adjacent sensors when working simultaneously. Results show that the average error in distance measurement using the ultrasonic sensor in laboratory conditions is ±0.53 cm. However, the increase of variability in field conditions reduces the accuracy of this kind of sensors when estimating distances to canopies. The average error in such situations is ±5.11 cm. When analyzing interferences of adjacent sensors 30 cm apart, the average error is ±17.46 cm. When sensors are separated 60 cm, the average error is ±9.29 cm. The ultrasonic sensor tested has been proven to be suitable to estimate distances to the canopy in field conditions when sensors are 60 cm apart or more and could, therefore, be used in a system to estimate structural canopy parameters in precision horticulture.

Relationship between vestibular hair cell loss and deficits in two anti-gravity reflexes in the rat.

The tail-lift reflex and the air-righting reflex in rats are anti-gravity reflexes that depend on vestibular function. To begin identifying their cellular basis, this study examined the relationship between reflex loss and the graded lesions caused in the vestibular sensory epithelia by varying doses of an ototoxic compound. After ototoxic exposure, we recorded these reflexes using high speed video. The movies were used to obtain objective measures of the reflexes: the minimum angle formed by the nose, the back of the neck and the base of the tail during the tail-lift maneuver and the time to right in the air-righting test. The vestibular sensory epithelia were then collected from the rats and used to estimate the loss of type I (HCI), type II (HCII) and all hair cells (HC) in both central and peripheral parts of the crista, utricle, and saccule. As expected, tail-lift angles decreased, and air-righting times increased, while the numbers of HCs remaining in the epithelia decreased in a dose-dependent manner. The results demonstrated greater sensitivity of HCI compared to HCII to the IDPN ototoxicity, as well as a relative resiliency of the saccule compared to the crista and utricle. Comparing the functional measures with the cell counts, we observed that loss of the tail-lift reflex associates better with HCI than with HCII loss. In contrast, most HCI in the crista and utricle were lost before air-righting times increased. These data suggest that these reflexes depend on the function of non-identical populations of vestibular HCs.

LFuji-air dataset: Annotated 3D LiDAR point clouds of Fuji apple trees for fruit detection scanned under different forced air flow conditions.

This article presents the LFuji-air dataset, which contains LiDAR based point clouds of 11 Fuji apples trees and the corresponding apples location ground truth. A mobile terrestrial laser scanner (MTLS) comprised of a LiDAR sensor and a real-time kinematics global navigation satellite system was used to acquire the data. The MTLS was mounted on an air-assisted sprayer used to generate different air flow conditions. A total of 8 scans per tree were performed, including scans from different LiDAR sensor positions (multi-view approach) and under different air flow conditions. These variability of the scanning conditions allows to use the LFuji-air dataset not only for training and testing new fruit detection algorithms, but also to study the usefulness of the multi-view approach and the application of forced air flow to reduce the number of fruit occlusions. The data provided in this article is related to the research article entitled "Fruit detection, yield prediction and canopy geometric characterization using LiDAR with forced air flow" [1].

Effects of co-exposure to CS2 and noise on hearing and balance in rats: continuous versus intermittent CS2 exposures.

BACKGROUND: Carbon disulfide (CS2) exacerbates the effect of noise on hearing, and disrupts the vestibular system. The goal of this study was to determine whether these effects are also observed with intermittent CS2 exposure. METHODS: Rats were exposed for 4 weeks (5 days/week, 6 h/day) to a band noise at 106 dB SPL either alone or combined with continuous (63 ppm or 250 ppm) or intermittent (15 min/h or 2 × 15 min/h at 250 ppm) CS2. Hearing function was assessed by measuring distortion product otoacoustic emissions (DPOAEs); balance was monitored based on the vestibulo-ocular reflex (VOR). Functional measurements were performed before, at the end of exposure and 4 weeks later. Histological analyses of the inner ear were also performed following exposure and after the 4-week recovery period. RESULTS: The results obtained here confirmed that CS2 exposure exerts two differential temporary effects on hearing: (1) it attenuates the noise-induced DPOAE decrease below 6 kHz probably through action on the middle ear reflex when exposure lasts 15 min per hour, and (2) continuous exposure to 250 ppm for 6 h extends the frequency range affected by noise up to 9.6 kHz (instead of 6 kHz with noise alone). With regard to balance, the VOR was reversibly disrupted at the two highest doses of CS2 (2 × 15 min/h and continuous 250 ppm). No morphological alterations to the inner ear were observed. CONCLUSION: These results reveal that short periods of CS2 exposure can alter the sensitivity of the cochlea to noise at a dose equivalent to only 10 times the short-term occupational limit value, and intermittent exposure to CS2 (2 × 15 min/h) can alter the function of the vestibular system.