Emerging Approaches for Restoration of Hearing and Vision.

Impairments of vision and hearing are highly prevalent conditions limiting the quality of life and presenting a major socioeconomic burden. For a long time, retinal and cochlear disorders have remained intractable for causal therapies, with sensory rehabilitation limited to glasses, hearing aids, and electrical cochlear or retinal implants. Recently, the application of gene therapy and optogenetics to eye and ear has generated hope for a fundamental improvement of vision and hearing restoration. To date, one gene therapy for the restoration of vision has been approved, and ongoing clinical trials will broaden its application including gene replacement, genome editing, and regenerative approaches. Moreover, optogenetics, i.e., controlling the activity of cells by light, offers a more general alternative strategy. Over little more than a decade, optogenetic approaches have been developed and applied to better understand the function of biological systems, while protein engineers have identified and designed new opsin variants with desired physiological features. Considering potential clinical applications of optogenetics, the spotlight is on the sensory systems, particularly the eye and ear. Multiple efforts have been undertaken to restore lost or hampered function in the eye and ear. Optogenetic stimulation promises to overcome fundamental shortcomings of electrical stimulation, namely, poor spatial resolution and cellular specificity, and accordingly to deliver more detailed sensory information. This review aims to provide a comprehensive reference on current gene therapeutic and optogenetic research relevant to the restoration of hearing and vision. We will introduce gene-therapeutic approaches and discuss the biotechnological and optoelectronic aspects of optogenetic hearing and vision restoration.

Multiscale photonic imaging of the native and implanted cochlea.

The cochlea of our auditory system is an intricate structure deeply embedded in the temporal bone. Compared with other sensory organs such as the eye, the cochlea has remained poorly accessible for investigation, for example, by imaging. This limitation also concerns the further development of technology for restoring hearing in the case of cochlear dysfunction, which requires quantitative information on spatial dimensions and the sensorineural status of the cochlea. Here, we employed X-ray phase-contrast tomography and light-sheet fluorescence microscopy and their combination for multiscale and multimodal imaging of cochlear morphology in species that serve as established animal models for auditory research. We provide a systematic reference for morphological parameters relevant for cochlear implant development for rodent and nonhuman primate models. We simulate the spread of light from the emitters of the optical implants within the reconstructed nonhuman primate cochlea, which indicates a spatially narrow optogenetic excitation of spiral ganglion neurons.

Endophilin-A regulates presynaptic Ca2+ influx and synaptic vesicle recycling in auditory hair cells.

Ribbon synapses of cochlear inner hair cells (IHCs) operate with high rates of neurotransmission; yet, the molecular regulation of synaptic vesicle (SV) recycling at these synapses remains poorly understood. Here, we studied the role of endophilins-A1-3, endocytic adaptors with curvature-sensing and curvature-generating properties, in mouse IHCs. Single-cell RT-PCR indicated the expression of endophilins-A1-3 in IHCs, and immunoblotting confirmed the presence of endophilin-A1 and endophilin-A2 in the cochlea. Patch-clamp recordings from endophilin-A-deficient IHCs revealed a reduction of Ca2+ influx and exocytosis, which we attribute to a decreased abundance of presynaptic Ca2+ channels and impaired SV replenishment. Slow endocytic membrane retrieval, thought to reflect clathrin-mediated endocytosis, was impaired. Otoferlin, essential for IHC exocytosis, co-immunoprecipitated with purified endophilin-A1 protein, suggestive of a molecular interaction that might aid exocytosis-endocytosis coupling. Electron microscopy revealed lower SV numbers, but an increased occurrence of coated structures and endosome-like vacuoles at IHC active zones. In summary, endophilins regulate Ca2+ influx and promote SV recycling in IHCs, likely via coupling exocytosis to endocytosis, and contributing to membrane retrieval and SV reformation.

Methods for multiscale structural and functional analysis of the mammalian cochlea.

The mammalian cochlea is a snail-shaped structure deeply that is embedded in the temporal bone and harbors the auditory sensory epithelium - the organ of Corti. Since the discovery of this remarkable hearing organ in the middle of the 19th century, generations of anatomists and physiologists have been attracted to study the structural and functional details of this intricate and delicate structure and thereby contributed to establishing our current understanding of peripheral sound encoding. Since these early days, the continued development of novel imaging technologies - both on light and electron microscopic level - has driven the auditory research field and now enables the visualization of cochlear structures across multiple scales with unprecedented clarity and exquisite detail. To honor these achievements, this review aims to provide a concise overview of current multi-scale imaging methodologies to investigate cochlear anatomy and cellular function in the peripheral auditory pathway. For this purpose, we will outline the technological concepts underlying these techniques - ranging from label-free to label-containing approaches - highlight their respective strengths and limitations and provide specific examples of their use in modern auditory research. We will focus on traditional as well as less conventional imaging techniques that present essential tools for unraveling the protein composition, nanoscale assembly, and physiology of the first auditory synapse and associated structures. In addition, we will introduce novel non-invasive large-scale methodologies that allow for high-resolution in situ imaging of the structurally-unperturbed cochlea and point out potential future applications. In combination, these techniques allow for a comprehensive multi-scale analysis of cochlear structure and function.

Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy.

Paraformaldehyde (PFA) is the most commonly used fixative for immunostaining of cells, but has been associated with various problems, ranging from loss of antigenicity to changes in morphology during fixation. We show here that the small dialdehyde glyoxal can successfully replace PFA Despite being less toxic than PFA, and, as most aldehydes, likely usable as a fixative, glyoxal has not yet been systematically tried in modern fluorescence microscopy. Here, we tested and optimized glyoxal fixation and surprisingly found it to be more efficient than PFA-based protocols. Glyoxal acted faster than PFA, cross-linked proteins more effectively, and improved the preservation of cellular morphology. We validated glyoxal fixation in multiple laboratories against different PFA-based protocols and confirmed that it enabled better immunostainings for a majority of the targets. Our data therefore support that glyoxal can be a valuable alternative to PFA for immunostaining.

A multicenter performance evaluation of the new Elecsys Vitamin D total III assay versus reference isotope dilution liquid chromatography tandem mass spectrometry and commercially available comparators.

BACKGROUND: Vitamin D deficiency/insufficiency and toxicity are worldwide issues; thus, accurate diagnostic assays are required to measure vitamin D. We evaluated the performance of the new Elecsys® Vitamin D total III assay (Roche Diagnostics International Ltd). METHODS: Repeatability and intermediate precision of the Elecsys Vitamin D total III assay (cobas e 601 analyzer) were evaluated at three sites using five human serum pools (HSPs) and two PreciControls (five-day model, one reagent lot [CLSI-EP05-A3]) and compared against prespecified acceptance criteria. A serum verification panel, with reference isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) values, was used for comparator assay/concordance studies at two sites, assessed using unweighted Deming regression. Testing of serum vs. plasma on the Elecsys assay was conducted at one site using samples from healthy adults; assessed using Passing-Bablok regression. RESULTS: Repeatability (HSP1 [16.8-18.4 ng/ml], SD 0.87-1.07; HSP5 [94.5-98.0 ng/ml], CV 1.58%-2.76%) and intermediate precision (HSP1, SD 1.14-1.77; HSP5, CV 2.00%-4.13%) met acceptance criteria across sites. Agreement was observed between the Elecsys assay and (i) the ID-LC-MS/MS verification panel (slope, 0.936-1.01; Pearson's r, 0.960-0.986) and (ii) comparator assays (slope, 0.921-1.15; Pearson's r, 0.958-0.982). The Elecsys assay correctly assigned the highest combined percentage of samples to deficient (100%) and insufficient (89.5%) vitamin D categories vs. comparator assays and demonstrated comparable performance in serum and plasma (y = 0.103 + 0.984x). CONCLUSIONS: The Elecsys Vitamin D total III assay demonstrated good analytical performance and compared favorably with other assays, supporting its use in clinical practice.

Mapping developmental maturation of inner hair cell ribbon synapses in the apical mouse cochlea.

Ribbon synapses of cochlear inner hair cells (IHCs) undergo molecular assembly and extensive functional and structural maturation before hearing onset. Here, we characterized the nanostructure of IHC synapses from late prenatal mouse embryo stages (embryonic days 14-18) into adulthood [postnatal day (P)48] using electron microscopy and tomography as well as optical nanoscopy of apical turn organs of Corti. We find that synaptic ribbon precursors arrive at presynaptic active zones (AZs) after afferent contacts have been established. These ribbon precursors contain the proteins RIBEYE and piccolino, tether synaptic vesicles and their delivery likely involves active, microtubule-based transport pathways. Synaptic contacts undergo a maturational transformation from multiple small to one single, large AZ. This maturation is characterized by the fusion of ribbon precursors with membrane-anchored ribbons that also appear to fuse with each other. Such fusion events are most frequently encountered around P12 and hence, coincide with hearing onset in mice. Thus, these events likely underlie the morphological and functional maturation of the AZ. Moreover, the postsynaptic densities appear to undergo a similar refinement alongside presynaptic maturation. Blockwise addition of ribbon material by fusion as found during AZ maturation might represent a general mechanism for modulating ribbon size.

Tryptophan-rich basic protein (WRB) mediates insertion of the tail-anchored protein otoferlin and is required for hair cell exocytosis and hearing.

The transmembrane recognition complex (TRC40) pathway mediates the insertion of tail-anchored (TA) proteins into membranes. Here, we demonstrate that otoferlin, a TA protein essential for hair cell exocytosis, is inserted into the endoplasmic reticulum (ER) via the TRC40 pathway. We mutated the TRC40 receptor tryptophan-rich basic protein (Wrb) in hair cells of zebrafish and mice and studied the impact of defective TA protein insertion. Wrb disruption reduced otoferlin levels in hair cells and impaired hearing, which could be restored in zebrafish by transgenic Wrb rescue and otoferlin overexpression. Wrb-deficient mouse inner hair cells (IHCs) displayed normal numbers of afferent synapses, Ca2+ channels, and membrane-proximal vesicles, but contained fewer ribbon-associated vesicles. Patch-clamp of IHCs revealed impaired synaptic vesicle replenishment. In vivo recordings from postsynaptic spiral ganglion neurons showed a use-dependent reduction in sound-evoked spiking, corroborating the notion of impaired IHC vesicle replenishment. A human mutation affecting the transmembrane domain of otoferlin impaired its ER targeting and caused an auditory synaptopathy. We conclude that the TRC40 pathway is critical for hearing and propose that otoferlin is an essential substrate of this pathway in hair cells.

Molecular Assembly and Structural Plasticity of Sensory Ribbon Synapses-A Presynaptic Perspective.

In the mammalian cochlea, specialized ribbon-type synapses between sensory inner hair cells (IHCs) and postsynaptic spiral ganglion neurons ensure the temporal precision and indefatigability of synaptic sound encoding. These high-through-put synapses are presynaptically characterized by an electron-dense projection-the synaptic ribbon-which provides structural scaffolding and tethers a large pool of synaptic vesicles. While advances have been made in recent years in deciphering the molecular anatomy and function of these specialized active zones, the developmental assembly of this presynaptic interaction hub remains largely elusive. In this review, we discuss the dynamic nature of IHC (pre-) synaptogenesis and highlight molecular key players as well as the transport pathways underlying this process. Since developmental assembly appears to be a highly dynamic process, we further ask if this structural plasticity might be maintained into adulthood, how this may influence the functional properties of a given IHC synapse and how such plasticity could be regulated on the molecular level. To do so, we take a closer look at other ribbon-bearing systems, such as retinal photoreceptors and pinealocytes and aim to infer conserved mechanisms that may mediate these phenomena.

The BEACH protein LRBA is required for hair bundle maintenance in cochlear hair cells and for hearing.

Lipopolysaccharide-responsive beige-like anchor protein (LRBA) belongs to the enigmatic class of BEACH domain-containing proteins, which have been attributed various cellular functions, typically involving intracellular protein and membrane transport processes. Here, we show that LRBA deficiency in mice leads to progressive sensorineural hearing loss. In LRBA knockout mice, inner and outer hair cell stereociliary bundles initially develop normally, but then partially degenerate during the second postnatal week. LRBA deficiency is associated with a reduced abundance of radixin and Nherf2, two adaptor proteins, which are important for the mechanical stability of the basal taper region of stereocilia. Our data suggest that due to the loss of structural integrity of the central parts of the hair bundle, the hair cell receptor potential is reduced, resulting in a loss of cochlear sensitivity and functional loss of the fraction of spiral ganglion neurons with low spontaneous firing rates. Clinical data obtained from two human patients with protein-truncating nonsense or frameshift mutations suggest that LRBA deficiency may likewise cause syndromic sensorineural hearing impairment in humans, albeit less severe than in our mouse model.

Unconventional molecular regulation of synaptic vesicle replenishment in cochlear inner hair cells.

Ribbon synapses of cochlear inner hair cells (IHCs) employ efficient vesicle replenishment to indefatigably encode sound. In neurons, neuroendocrine and immune cells, vesicle replenishment depends on proteins of the mammalian uncoordinated 13 (Munc13, also known as Unc13) and Ca(2+)-dependent activator proteins for secretion (CAPS) families, which prime vesicles for exocytosis. Here, we tested whether Munc13 and CAPS proteins also regulate exocytosis in mouse IHCs by combining immunohistochemistry with auditory systems physiology and IHC patch-clamp recordings of exocytosis in mice lacking Munc13 and CAPS isoforms. Surprisingly, we did not detect Munc13 or CAPS proteins at IHC presynaptic active zones and found normal IHC exocytosis as well as auditory brainstem responses (ABRs) in Munc13 and CAPS deletion mutants. Instead, we show that otoferlin, a C2-domain protein that is crucial for vesicular fusion and replenishment in IHCs, clusters at the plasma membrane of the presynaptic active zone. Electron tomography of otoferlin-deficient IHC synapses revealed a reduction of short tethers holding vesicles at the active zone, which might be a structural correlate of impaired vesicle priming in otoferlin-deficient IHCs. We conclude that IHCs use an unconventional priming machinery that involves otoferlin.

Deletion of interleukin-6 signal transducer gp130 in small sensory neurons attenuates mechanonociception and down-regulates TRPA1 expression.

Glycoprotein 130 (gp130) is the signal transducing receptor subunit for cytokines of the interleukin-6 (IL-6) family, and it is expressed in a multitude of cell types of the immune and nervous system. IL-6-like cytokines are not only key regulators of innate immunity and inflammation but are also essential factors for the differentiation and development of the somatosensory system. Mice with a null mutation of gp130 in primary nociceptive afferents (SNS-gp130(-/-)) are largely protected from hypersensitivity to mechanical stimuli in mouse models of pathological pain. Therefore, we set out to investigate how neuronal gp130 regulates mechanonociception. SNS-gp130(-/-) mice revealed reduced mechanosensitivity to high mechanical forces in the von Frey assay in vivo, and this was associated with a reduced sensitivity of nociceptive primary afferents in vitro. Together with these findings, transient receptor potential ankyrin 1 (TRPA1) mRNA expression was significantly reduced in DRG from SNS-gp130(-/-) mice. This was also reflected by a reduced number of neurons responding with calcium transients to TRPA1 agonists in primary DRG cultures. Downregulation of Trpa1 expression was predominantly discovered in nonpeptidergic neurons, with the deficit becoming evident during stages of early postnatal development. Regulation of Trpa1 mRNA expression levels downstream of gp130 involved the classical Janus kinase family-signal transducer and activator of transcription pathway. Our results closely link proinflammatory cytokines to the expression of TRPA1, both of which have been shown to contribute to hypersensitive pain states. We suggest that gp130 has an essential role in mechanonociception and in the regulation of TRPA1 expression.

Synaptic vesicle glycoprotein 2A modulates vesicular release and calcium channel function at peripheral sympathetic synapses.

Synaptic vesicle glycoprotein (SV)2A is a transmembrane protein found in secretory vesicles and is critical for Ca(2+) -dependent exocytosis in central neurons, although its mechanism of action remains uncertain. Previous studies have proposed, variously, a role of SV2 in the maintenance and formation of the readily releasable pool (RRP) or in the regulation of Ca(2+) responsiveness of primed vesicles. Such previous studies have typically used genetic approaches to ablate SV2 levels; here, we used a strategy involving small interference RNA (siRNA) injection to knockdown solely presynaptic SV2A levels in rat superior cervical ganglion (SCG) neuron synapses. Moreover, we investigated the effects of SV2A knockdown on voltage-dependent Ca(2+) channel (VDCC) function in SCG neurons. Thus, we extended the studies of SV2A mechanisms by investigating the effects on vesicular transmitter release and VDCC function in peripheral sympathetic neurons. We first demonstrated an siRNA-mediated SV2A knockdown. We showed that this SV2A knockdown markedly affected presynaptic function, causing an attenuated RRP size, increased paired-pulse depression and delayed RRP recovery after stimulus-dependent depletion. We further demonstrated that the SV2A-siRNA-mediated effects on vesicular release were accompanied by a reduction in VDCC current density in isolated SCG neurons. Together, our data showed that SV2A is required for correct transmitter release at sympathetic neurons. Mechanistically, we demonstrated that presynaptic SV2A: (i) acted to direct normal synaptic transmission by maintaining RRP size, (ii) had a facilitatory role in recovery from synaptic depression, and that (iii) SV2A deficits were associated with aberrant Ca(2+) current density, which may contribute to the secretory phenotype in sympathetic peripheral neurons.

Automated Competitive Protein-Binding Assay for Total 25-OH Vitamin D, Multicenter Evaluation and Practical Performance.

BACKGROUND: The Roche Elecsys Vitamin D Total competitive protein-binding assay uses recombinant vitamin D binding protein for measuring 25-hydroxyvitamin D (25-OHD), which is different from commonly used antibody assays. METHODS: The assay, standardized against LC-MS/MS, was tested at four sites. Evaluation included precision; between-laboratory variability; functional sensitivity; correlation to LC-MS/MS, HPLC, and immunoassays; as well as robustness, traceability, and EQAS performance. RESULTS: Precision testing showed within-run coefficient of variations (CVs) of ≤ 7%, within-laboratory CVs of <9.5%, between-laboratory precision CVs of ≤ 10.1%, and a functional sensitivity below 9.8 nmol/l (at CV 12.9%). The assay showed equivalent 25-OHD levels for matched serum and plasma samples, good reagent lot-to-lot consistency in pooled sera over time, and good agreement with HPLC (relative bias -8.8%). Comparison with LC-MS/MS methods yielded relative biases of -15.4, -13.5, -10.2, and 3.2%. Comparison against immunoassays showed a relative bias of 14.5% (DiaSorin Liaison) and -58.2% (IDS-iSYS). The overall mean results in 2 years DEQAS was 102% of the ALTM. In a certified reference patient panel, the average bias was < 4% for the sum of 25-OHD2 and 25-OHD3. CONCLUSION: The Elecsys Vitamin D Total assay demonstrated good overall performance and is, according to present standards, very suitable for automated measurement of 25-OHD.

Slow kinesin-dependent microtubular transport facilitates ribbon synapse assembly in developing cochlear inner hair cells.

Sensory synapses are characterized by electron-dense presynaptic specializations, so-called synaptic ribbons. In cochlear inner hair cells (IHCs), ribbons play an essential role as core active zone (AZ) organizers, where they tether synaptic vesicles, cluster calcium channels and facilitate the temporally-precise release of primed vesicles. While a multitude of studies aimed to elucidate the molecular composition and function of IHC ribbon synapses, the developmental formation of these signalling complexes remains largely elusive to date. To address this shortcoming, we performed long-term live-cell imaging of fluorescently-labelled ribbon precursors in young postnatal IHCs to track ribbon precursor motion. We show that ribbon precursors utilize the apico-basal microtubular (MT) cytoskeleton for targeted trafficking to the presynapse, in a process reminiscent of slow axonal transport in neurons. During translocation, precursor volume regulation is achieved by highly dynamic structural plasticity - characterized by regularly-occurring fusion and fission events. Pharmacological MT destabilization negatively impacted on precursor translocation and attenuated structural plasticity, whereas genetic disruption of the anterograde molecular motor Kif1a impaired ribbon volume accumulation during developmental maturation. Combined, our data thus indicate an essential role of the MT cytoskeleton and Kif1a in adequate ribbon synapse formation and structural maintenance.

Alternative Food Networks in Latin America-exploring PGS (Participatory Guarantee Systems) markets and their consumers: a cross-country comparison.

Alternative food networks (AFN) are argued to provide platforms to re-socialize and re-spacealize food, establish and contribute to democratic participation in local food chains, and foster producer-consumer relations and trust. As one of the most recent examples of AFN, Participatory Guarantee Systems (PGS) have gained notable traction in attempting to redefine consumer-producer relations in the organic value chain. The participation of stakeholders, such as consumers, has been a key element theoretically differentiating PGS from other organic verification systems. While research on farmer participation in PGS is attracting interest, consumer participation is still widely overlooked. Using a mixed methods approach, this paper describes five PGS markets in Mexico, Chile and Bolivia. A survey was conducted with consumers in the PGS markets to explore their awareness of the PGS, how consumers participate in the PGS, and their level of trust in the respective PGS and its certified products. Results showed a low level of awareness of PGS among market consumers, few participation possibilities, and minimal consumer participation overall. Nevertheless, trust in organic quality was generally high. Consumers primarily relied on the direct relationship with producers and the PGS market itself as sources of trust. These results provide novel insight into PGS consumer-market interactions, and contribute to discussions concerning social embeddedness, awareness and participation within AFN.

Age-dependent structural reorganization of utricular ribbon synapses.

In mammals, spatial orientation is synaptically-encoded by sensory hair cells of the vestibular labyrinth. Vestibular hair cells (VHCs) harbor synaptic ribbons at their presynaptic active zones (AZs), which play a critical role in molecular scaffolding and facilitate synaptic release and vesicular replenishment. With advancing age, the prevalence of vestibular deficits increases; yet, the underlying mechanisms are not well understood and the possible accompanying morphological changes in the VHC synapses have not yet been systematically examined. We investigated the effects of maturation and aging on the ultrastructure of the ribbon-type AZs in murine utricles using various electron microscopic techniques and combined them with confocal and super-resolution light microscopy as well as metabolic imaging up to 1 year of age. In older animals, we detected predominantly in type I VHCs the formation of floating ribbon clusters, mostly consisting of newly synthesized ribbon material. Our findings suggest that VHC ribbon-type AZs undergo dramatic structural alterations upon aging.

The synaptic vesicle glycoprotein 2A ligand levetiracetam inhibits presynaptic Ca2+ channels through an intracellular pathway.

Levetiracetam (LEV) is a prominent antiepileptic drug that binds to neuronal synaptic vesicle glycoprotein 2A protein and has reported effects on ion channels, but with a poorly defined mechanism of action. We investigated inhibition of voltage-dependent Ca(2+) (Ca(V)) channels as a potential mechanism through which LEV exerts effects on neuronal activity. We used electrophysiological methods to investigate the effects of LEV on cholinergic synaptic transmission and Ca(V) channel activity in superior cervical ganglion neurons (SCGNs). In parallel, we investigated the effects of the inactive LEV R-enantiomer, (R)-α-ethyl-2-oxo-1-pyrrolidine acetamide (UCB L060). LEV but not UCB L060 (each at 100 µM) inhibited synaptic transmission between SCGNs in long-term culture in a time-dependent manner, significantly reducing excitatory postsynaptic potentials after a ≥30-min application. In isolated SCGNs, LEV pretreatment (≥1 h) but not short-term application (5 min) significantly inhibited whole-cell Ba(2+) current (I(Ba)) amplitude. In current-clamp recordings, LEV reduced the amplitude of the afterhyperpolarizing potential in a Ca(2+)-dependent manner but also increased the action potential latency in a Ca(2+)-independent manner, which suggests additional mechanisms associated with reduced excitability. Intracellular LEV application (4-5 min) caused rapid inhibition of I(Ba) amplitude, to an extent comparable to that seen with extracellular LEV pretreatment (≥1 h). Neither pretreatment nor intracellular application of UCB L060 produced any inhibitory effects on I(Ba) amplitude. These results identify a stereospecific intracellular pathway through which LEV inhibits presynaptic Ca(V) channels; resultant reductions in neuronal excitability are proposed to contribute to the anticonvulsant effects of LEV.

Indigenous farmers' perceptions of problems in the rice field agroecosystems in the upper Baram, Malaysia.

BACKGROUND: Rice field agroecosystems produce food for more than half of the world's population and deliver important services supporting farmers' livelihoods. However, traditional rice field agroecosystems are facing a variety of problems, including pests or markets that are hard to access. This research explored indigenous farmers' perceptions of the problems, their causes and consequences, and the solutions applied to address them in the rice field agroecosystem. Furthermore, the study investigated how indigenous farmers related these problems to the surrounding landscape elements and to microzones in the fields. METHODS: Data were collected in two villages in the upper Baram, Sarawak using a qualitative approach that included sketch drawings and face-to-face interviews. Forty-three indigenous farmers of the Kenyah, Penan and Sa'ban ethnic groups were interviewed in their rice fields. The sketch drawings were used to identify the perceived landscape elements, while the oral interviews were employed to identify perceived microzones. Furthermore, the interviews elicited the perceived problems in the rice field agroecosystem and their relations to landscape elements and microzones. RESULTS: The findings identified a total of nine environmental problems, e.g. animal disturbance, six social problems, e.g. difficult to access farm inputs, and eight agricultural technology system problems, e.g. poor soil quality, with some found to be rooted in complex causes and affecting agricultural productivity. While some problems were perceived at field level, microzones were frequently used as sub-field indicators of the problems. The surrounding landscape elements were perceived as both a source of the problems and as a means of avoiding them. To solve the problems, farmers applied preventive and reactive strategies based on traditional knowledge and scientific knowledge, resulting in a hybridisation of knowledge systems. CONCLUSIONS: By including environmental, social, agricultural technology system problems and different spatial scales, this research contributes to addressing issues that can be overlooked when focusing on only one dimension of the problems. These results contribute to a better understanding of how indigenous farmers perceive, cope with and adapt to problems in rice field agroecosystems, which is important for landscape management.

Monoallelic loss of the F-actin-binding protein radixin facilitates startle reactivity and pre-pulse inhibition in mice.

Hearing impairment is one of the most common disorders with a global burden and increasing prevalence in an ever-aging population. Previous research has largely focused on peripheral sensory perception, while the brain circuits of auditory processing and integration remain poorly understood. Mutations in the rdx gene, encoding the F-actin binding protein radixin (Rdx), can induce hearing loss in human patients and homozygous depletion of Rdx causes deafness in mice. However, the precise physiological function of Rdx in hearing and auditory information processing is still ill-defined. Here, we investigated consequences of rdx monoallelic loss in the mouse. Unlike the homozygous (-/-) rdx knockout, which is characterized by the degeneration of actin-based stereocilia and subsequent hearing loss, our analysis of heterozygous (+/-) mutants has revealed a different phenotype. Specifically, monoallelic loss of rdx potentiated the startle reflex in response to acoustic stimulation of increasing intensities, suggesting a gain of function relative to wildtype littermates. The monoallelic loss of the rdx gene also facilitated pre-pulse inhibition of the acoustic startle reflex induced by weak auditory pre-pulse stimuli, indicating a modification to the circuit underlying sensorimotor gating of auditory input. However, the auditory brainstem response (ABR)-based hearing thresholds revealed a mild impairment in peripheral sound perception in rdx (+/-) mice, suggesting minor aberration of stereocilia structural integrity. Taken together, our data suggest a critical role of Rdx in the top-down processing and/or integration of auditory signals, and therefore a novel perspective to uncover further Rdx-mediated mechanisms in central auditory information processing.