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.

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.

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.

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.

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.

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.

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.

What can we learn from past and recent Bavarian knowledge for the future development of European veterinary herbal medicine? An ethnoveterinary study.

ETHNOPHARMACOLOGICAL RELEVANCE: While the interest in finding medical solutions for the worldwide antibiotics crisis is rising, the legal possibility of simplified authorization of herbal veterinary medicinal products is dwindling. An important basis for both the preservation and development of knowledge in veterinary herbal medicine are pharmacological and clinical studies on the performance of herbal remedies, based on historical written sources on the treatment of farm animals with medicinal plants, as well as current ethnoveterinary research. Nevertheless, there is only limited systematic ethnoveterinary research in Europe, with the exceptions of the Mediterranean region, Switzerland and Austria. We conducted a survey on the ethnoveterinary knowledge of farmers in Bavaria, and analyzed two regional historical textbooks. AIM OF THE STUDY: We documented the local veterinary knowledge about livestock in Bavaria based upon local historical textbooks and upon ethnoveterinary interviews to discover opportunities for the future development of European veterinary herbal medicine. MATERIAL AND METHODS: In 2018/2019 we conducted 77 semi-structured interviews with 101 farmers from different types of farms. Detailed information about homemade herbal remedies (plant species, plant part, manufacturing process, source of knowledge) and the corresponding use reports (target animal species, category of use, route of administration, dosage, source of knowledge, frequency of use, last time of use and farmers' satisfaction) were collected. To compare our data with the literature, the use reports of two local historical textbooks were analyzed and compared with the data from the interviews. RESULTS: 716 homemade remedy reports (HRs) for altogether 884 use reports (URs) were documented in this study. We picked the 363 HRs that consisted of a single plant species with or without other natural products (HSHRs) for a deeper analysis. These HSHRs were prepared from 108 plant species that belonged to 57 botanical families. The most URs were documented for the families of: Asteraceae, Linaceae and Urticaceae. Calendula officinalis L. (Asteraceae), Linum usitatissimum L. (Linaceae) and Urtica dioica L. (Urticaceae) were the most often documented single species. A total of 448 URs were gathered for the 363 HSHRs. The largest number of URs was for treatments of gastrointestinal disorders and metabolic dysfunctions, followed by skin alterations and sores. For nearly half of the URs the source of knowledge was family and friends. For 80 URs the source of knowledge was different from that of the corresponding HSHRs. For 68% of the URs farmers mentioned at least one use during the last 5 years. Half of the plant species that were mentioned in the historical literature were also mentioned in URs by the interviewees. CONCLUSION: In Bavaria, medicinal plants are actively used by farmers to treat their livestock with a high level of satisfaction. The knowledge is not passed on from generation to generation in a purely static way, but is dynamically developed by the users in almost one fifth of the URs. Ethnoveterinary research combined with data from regional historical textbooks may facilitate pharmacological and clinical studies in veterinary medicine, and the discussion about a simplified registration for traditional herbal veterinary medicinal products.

Local plant knowledge and its variation among farmer's families in the Napf region, Switzerland.

BACKGROUND: Local plant knowledge typically is unevenly distributed within a community. This knowledge variation is important in understanding people's relationship with their environment. Here we ask about knowledge variation among farmers' families in the Napf region of Switzerland. METHODS: In 2008 and 2009, 60 adults and children living on 14 farms were interviewed about known and used plant species, and the data analyzed for knowledge variation. The farms were chosen by random stratified sampling, and freelisting and semi-structured interviews were conducted individually in the local idiom. The data were organized in an access database and analyzed with descriptive statistics, correlations, Mann-Whitney U tests and cultural domain analysis. RESULTS: Totally, 456 folk taxa were listed, whereas frequently listed species are common meadow and forest species. Uses were indicated for 391 taxa, most of them culinary, followed by fodder, wood, medicinal and ornamental uses. Local plant knowledge correlates with age and gender. Due to professional specialization, adults above 20 years have broader plant knowledge than children and adolescents. This is true for almost all examined habitat and plant use categories except for toy uses. Women and men share a common body of plant knowledge especially about herbaceous grassland species and woody species. Specialized knowledge of men is linked to cattle fodder and the processing of wood, specialized knowledge of women concerns edible, medicinal and ornamental plants, often garden species, but also herbaceous forest species. CONCLUSION: In a rural region like the Napf, people retain a solid basis of plant knowledge. The variation of plant knowledge within farmers' families of this region reflects sociocultural patterns. As these patterns are changing and as (agro)biodiversity is declining, local plant knowledge in the Napf region is suspected to undergo a mainstreaming process.

RIM-Binding Proteins Are Required for Normal Sound-Encoding at Afferent Inner Hair Cell Synapses.

The afferent synapses between inner hair cells (IHC) and spiral ganglion neurons are specialized to faithfully encode sound with sub-millisecond precision over prolonged periods of time. Here, we studied the role of Rab3 interacting molecule-binding proteins (RIM-BP) 1 and 2 - multidomain proteins of the active zone known to directly interact with RIMs, Bassoon and Ca V 1.3 - in IHC presynaptic function and hearing. Recordings of auditory brainstem responses and otoacoustic emissions revealed that genetic disruption of RIM-BPs 1 and 2 in mice (RIM-BP1/2-/- ) causes a synaptopathic hearing impairment exceeding that found in mice lacking RIM-BP2 (RIM-BP2-/- ). Patch-clamp recordings from RIM-BP1/2-/- IHCs indicated a subtle impairment of exocytosis from the readily releasable pool of synaptic vesicles that had not been observed in RIM-BP2-/- IHCs. In contrast, the reduction of Ca2+-influx and sustained exocytosis was similar to that in RIMBP2-/- IHCs. We conclude that both RIM-BPs are required for normal sound encoding at the IHC synapse, whereby RIM-BP2 seems to take the leading role.

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.

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.

Multichannel optogenetic stimulation of the auditory pathway using microfabricated LED cochlear implants in rodents.

When hearing fails, electrical cochlear implants (eCIs) provide the brain with auditory information. One important bottleneck of CIs is the poor spectral selectivity that results from the wide current spread from each of the electrode contacts. Optical CIs (oCIs) promise to make better use of the tonotopic order of spiral ganglion neurons (SGNs) inside the cochlea by spatially confined stimulation. Here, we established multichannel oCIs based on light-emitting diode (LED) arrays and used them for optical stimulation of channelrhodopsin (ChR)-expressing SGNs in rodents. Power-efficient blue LED chips were integrated onto microfabricated 15-µm-thin polyimide-based carriers comprising interconnecting lines to address individual LEDs by a stationary or mobile driver circuitry. We extensively characterized the optoelectronic, thermal, and mechanical properties of the oCIs and demonstrated stability over weeks in vitro. We then implanted the oCIs into ChR-expressing rats and gerbils, and characterized multichannel optogenetic SGN stimulation by electrophysiological and behavioral experiments. Improved spectral selectivity was directly demonstrated by recordings from the auditory midbrain. Long-term experiments in deafened ChR-expressing rats and in nontreated control animals demonstrated specificity of optogenetic stimulation. Behavioral studies on animals carrying a wireless oCI sound processor revealed auditory percepts. This study demonstrates hearing restoration with improved spectral selectivity by an LED-based multichannel oCI system.

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.

Overloaded Adeno-Associated Virus as a Novel Gene Therapeutic Tool for Otoferlin-Related Deafness.

Hearing impairment is the most common sensory disorder in humans. So far, rehabilitation of profoundly deaf subjects relies on direct stimulation of the auditory nerve through cochlear implants. However, in some forms of genetic hearing impairment, the organ of Corti is structurally intact and therapeutic replacement of the mutated gene could potentially restore near natural hearing. In the case of defects of the otoferlin gene (OTOF), such gene therapy is hindered by the size of the coding sequence (~6 kb) exceeding the cargo capacity (<5 kb) of the preferred viral vector, adeno-associated virus (AAV). Recently, a dual-AAV approach was used to partially restore hearing in deaf otoferlin knock-out (Otof-KO) mice. Here, we employed in vitro and in vivo approaches to assess the gene-therapeutic potential of naturally-occurring and newly-developed synthetic AAVs overloaded with the full-length Otof coding sequence. Upon early postnatal injection into the cochlea of Otof-KO mice, overloaded AAVs drove specific expression of otoferlin in ~30% of all IHCs, as demonstrated by immunofluorescence labeling and polymerase chain reaction. Recordings of auditory brainstem responses and a behavioral assay demonstrated partial restoration of hearing. Together, our results suggest that viral gene therapy of DFNB9-using a single overloaded AAV vector-is indeed feasible, reducing the complexity of gene transfer compared to dual-AAV approaches.

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.

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.

The changing face of farmers' home gardens: a diachronic analysis from Sillian (Eastern Tyrol, Austria).

BACKGROUND: Home gardens are an integral part of many traditional land use systems around the world. They are subject to various conversion processes and undergo a variety of changes. We were interested if change is an ongoing process in farmers' home gardens of Eastern Tyrol (Austria). METHODS: In Sillian, 16 farmers' home gardens (FHGs) were studied. They had been studied in 1998 and were revisited in 2013 including again a botanical inventory of cultivated and non-cultivated plants, and structured interviews on appearance, management and plant use. In 2017, all the 16 gardens were visited again to verify whether any visible change on spatial configuration had occurred. RESULTS: The home garden size had decreased between 1998 and 2013. A wider range of sizes was observed. The occurrence of plant taxa per garden was the same but an increase in the standard deviation of occurrence is seen. Plant diversity (occ./m2) increased between 1998 and 2013. Seventy-nine plant taxa were no longer cultivated in 2013, but 95 new plant taxa were being cultivated. The correlation between garden size and occurrence was not significant, i.e. small gardens might host many different plant taxa or large gardens might have fewer plant taxa. The occurrence for certain use categories was not significantly different between the years, except for the increase in the occurrence of plant taxa used as food and the food subcategory spice. The mean abundance of individuals for all plant taxa showed a significant decrease between the years. In 2013, an increase in standard deviation of abundance is seen. The variation in the different use categories expressed in abundance between the years was not significantly different, except for the decrease in the abundance of plant taxa used as food. Between 1998 and 2017, six home gardens showed a change of their spatial configuration (replacement by raised beds; merging with other structures; conversion to lawn). One FHG shows signs of abandonment. CONCLUSIONS: In Sillian, gardens are by no way static agroecological units, but are dynamic and individual in their appearance, composition and function. Farmers' home gardens in Sillian show a trend towards becoming more individual, i.e. conversion from being a product of a homogenous local cultural script of the community into an area where gardeners define more individually the role that farmers' homegardens are expected to play for them or their family.

Balancing presynaptic release and endocytic membrane retrieval at hair cell ribbon synapses.

The timely and reliable processing of auditory and vestibular information within the inner ear requires highly sophisticated sensory transduction pathways. On a cellular level, these demands are met by hair cells, which respond to sound waves - or alterations in body positioning - by releasing glutamate-filled synaptic vesicles (SVs) from their presynaptic active zones with unprecedented speed and exquisite temporal fidelity, thereby initiating the auditory and vestibular pathways. In order to achieve this, hair cells have developed anatomical and molecular specializations, such as the characteristic and name-giving 'synaptic ribbons' - presynaptically anchored dense bodies that tether SVs prior to release - as well as other unique or unconventional synaptic proteins. The tightly orchestrated interplay between these molecular components enables not only ultrafast exocytosis, but similarly rapid and efficient compensatory endocytosis. So far, the knowledge of how endocytosis operates at hair cell ribbon synapses is limited. In this Review, we summarize recent advances in our understanding of the SV cycle and molecular anatomy of hair cell ribbon synapses, with a focus on cochlear inner hair cells.

Cytomatrix proteins CAST and ELKS regulate retinal photoreceptor development and maintenance.

At the presynaptic active zone (AZ), the related cytomatrix proteins CAST and ELKS organize the presynaptic release machinery. While CAST is known to regulate AZ size and neurotransmitter release, the role of ELKS and the integral system of CAST/ELKS together is poorly understood. Here, we show that CAST and ELKS have both redundant and unique roles in coordinating synaptic development, function, and maintenance of retinal photoreceptor ribbon synapses. A CAST/ELKS double knockout (dKO) mouse showed high levels of ectopic synapses and reduced responses to visual stimulation. Ectopic formation was not observed in ELKS conditional KO but progressively increased with age in CAST KO mice with higher rates in the dKO. Presynaptic calcium influx was strongly reduced in rod photoreceptors of CAST KO and dKO mice. Three-dimensional scanning EM reconstructions showed structural abnormalities in rod triads of CAST KO and dKO. Remarkably, AAV-mediated acute ELKS deletion after synapse maturation induced neurodegeneration and loss of ribbon synapses. These results suggest that CAST and ELKS work in concert to promote retinal synapse formation, transmission, and maintenance.