Reliability of the 2021 National Youth Risk Behavior Survey Questionnaire.

PURPOSE: The Youth Risk Behavior Survey (YRBS) monitors behaviors, experiences, and conditions affecting the health of high school students nationwide. This study examined the test-retest reliability of the 2021 national YRBS questionnaire. DESIGN: Respondents completed a Time 1 and Time 2 paper-and-pencil questionnaire approximately 2 weeks apart during February to May 2022. Data were linked in such a way as to preserve anonymity. SETTING: Convenience sample of high schools. SUBJECTS: High school students (N = 588). MEASURES: Health risk behaviors and experiences assessed on the 2021 national YRBS questionnaire. ANALYSIS: Time 1 and Time 2 responses were compared for each questionnaire item using the McNemar's test. Then, Cohen's kappa coefficients tested the agreement between Time 1 and Time 2 responses overall, and by sex, grade, and Black, White, and Hispanic race and ethnicity. RESULTS: Among the 74 items analyzed, 96% had at least moderate reliability, and 73% had substantial or almost perfect reliability. The mean Cohen's kappa was .68. McNemar's test findings showed Time 1 and Time 2 data significantly differed (P < .01) for 9 items (12%). CONCLUSION: Reliable health behavior measures are important in the development of youth-focused public health programs and policies. Findings suggest the national YRBS questionnaire is a reliable instrument. Such findings lend support to relying on adolescent self-reported data when monitoring health behaviors using the YRBS.

Overview and Methods for the Youth Risk Behavior Surveillance System - United States, 2021.

The Youth Risk Behavior Surveillance System (YRBSS) is the largest public health surveillance system in the United States, monitoring a broad range of health-related behaviors among high school students. The system includes a nationally representative Youth Risk Behavior Survey (YRBS) and separate school-based YRBSs conducted by states, tribes, territories, and local school districts. In 2021, these surveys were conducted during the COVID-19 pandemic. The pandemic underscored the importance of data in understanding changes in youth risk behaviors and addressing the multifaceted public health needs of youths. This overview report describes 2021 YRBSS survey methodology, including sampling, data collection procedures, response rates, data processing, weighting, and analyses. The 2021 YRBS participation map, survey response rates, and a detailed examination of student demographic characteristics are included in this report. During 2021, in addition to the national YRBS, a total of 78 surveys were administered to high school students across the United States, representing the national population, 45 states, two tribal governments, three territories, and 28 local school districts. YRBSS data from 2021 provided the first opportunity since the onset of the COVID-19 pandemic to compare youth health behaviors using long-term public health surveillance. Approximately half of all student respondents represented racial and ethnic minority groups, and approximately one in four identified as lesbian, gay, bisexual, questioning, or other (a sexual identity other than heterosexual) (LGBQ+). These findings reflect shifts in youth demographics, with increased percentages of racial and ethnic minority and LGBQ+ youths compared with previous YRBSS cycles. Educators, parents, local decision makers, and other partners use YRBSS data to monitor health behavior trends, guide school health programs, and develop local and state policy. These and future data can be used in developing health equity strategies to address long-term disparities so that all youths can thrive in safe and supportive environments. This overview and methods report is one of 11 featured in this MMWR supplement. Each report is based on data collected using methods presented in this overview. A full description of YRBSS results and downloadable data are available (https://www.cdc.gov/healthyyouth/data/yrbs/index.htm).

Overview and Methodology of the Adolescent Behaviors and Experiences Survey - United States, January-June 2021.

Many U.S. schools closed nationwide in March 2020 to prevent the spread of COVID-19. School closures and online-only instruction have negatively affected certain students, with studies showing adverse effects of the pandemic on mental health. However, little is known about other experiences such as economic and food insecurity and abuse by a parent, as well as risk behaviors such as alcohol and drug use among youths across the United States during the pandemic. To address this gap, CDC developed the one-time, online Adolescent Behaviors and Experiences Survey (ABES), which was conducted during January-June 2021 to assess student behaviors and experiences during the COVID-19 pandemic among high school students, including unintentional injury, violence, tobacco product use, sexual behaviors, and dietary behaviors. This overview report of the ABES MMWR Supplement describes the ABES methodology, including the student questionnaire and administration, sampling, data collection, weighting, and analysis. ABES used a stratified, three-stage cluster probability-based sampling approach to obtain a nationally representative sample of students in grades 9-12 attending public and private schools. Teachers of selected classes provided students with access to the anonymous online survey while following local consent procedures. Data were collected using a 110-item questionnaire during January-June 2021 in 128 schools. A total of 7,998 students submitted surveys, and 7,705 of these surveys had valid data (i.e., ≥20 questions answered). The school response rate was 38%, the student response rate was 48%, and the overall response rate was 18%. Information on mode of instruction and school-provided equipment was also collected from all sampled schools. This overview report provides student- and school-level characteristics obtained from descriptive analyses, and the other reports in the ABES MMWR Supplement include information on substance use, mental health and suicidality, perceived racism, and disruptions to student life among high school students. Findings from ABES during the COVID-19 pandemic can help guide parents, teachers, school administrators, community leaders, clinicians, and public health officials in decision-making for student support and school health programs.

Nuclear crowding and nonlinear diffusion during interkinetic nuclear migration in the zebrafish retina.

An important question in early neural development is the origin of stochastic nuclear movement between apical and basal surfaces of neuroepithelia during interkinetic nuclear migration. Tracking of nuclear subpopulations has shown evidence of diffusion - mean squared displacements growing linearly in time - and suggested crowding from cell division at the apical surface drives basalward motion. Yet, this hypothesis has not yet been tested, and the forces involved not quantified. We employ long-term, rapid light-sheet and two-photon imaging of early zebrafish retinogenesis to track entire populations of nuclei within the tissue. The time-varying concentration profiles show clear evidence of crowding as nuclei reach close-packing and are quantitatively described by a nonlinear diffusion model. Considerations of nuclear motion constrained inside the enveloping cell membrane show that concentration-dependent stochastic forces inside cells, compatible in magnitude to those found in cytoskeletal transport, can explain the observed magnitude of the diffusion constant.

Overview and Methods for the Youth Risk Behavior Surveillance System - United States, 2019.

Health risk behaviors practiced during adolescence often persist into adulthood and contribute to the leading causes of morbidity and mortality in the United States. Youth health behavior data at the national, state, territorial, tribal, and local levels help monitor the effectiveness of public health interventions designed to promote adolescent health. The Youth Risk Behavior Surveillance System (YRBSS) is the largest public health surveillance system in the United States, monitoring a broad range of health-related behaviors among high school students. YRBSS includes a nationally representative Youth Risk Behavior Survey (YRBS) and separate state, local school district, territorial, and tribal school-based YRBSs. This overview report describes the surveillance system and the 2019 survey methodology, including sampling, data collection procedures, response rates, data processing, weighting, and analyses presented in this MMWR Supplement. A 2019 YRBS participation map, survey response rates, and student demographic characteristics are included. In 2019, a total of 78 YRBSs were administered to high school student populations across the United States (national and 44 states, 28 local school districts, three territories, and two tribal governments), the greatest number of participating sites with representative data since the surveillance system was established in 1991. The nine reports in this MMWR Supplement are based on national YRBS data collected during August 2018-June 2019. A full description of 2019 YRBS results and downloadable data are available (https://www.cdc.gov/healthyyouth/data/yrbs/index.htm).Efforts to improve YRBSS and related data are ongoing and include updating reliability testing for the national questionnaire, transitioning to electronic survey administration (e.g., pilot testing for a tablet platform), and exploring innovative analytic methods to stratify data by school-level socioeconomic status and geographic location. Stakeholders and public health practitioners can use YRBS data (comparable across national, state, tribal, territorial, and local jurisdictions) to estimate the prevalence of health-related behaviors among different student groups, identify student risk behaviors, monitor health behavior trends, guide public health interventions, and track progress toward national health objectives.

On-Site Ribosome Remodeling by Locally Synthesized Ribosomal Proteins in Axons.

Ribosome assembly occurs mainly in the nucleolus, yet recent studies have revealed robust enrichment and translation of mRNAs encoding many ribosomal proteins (RPs) in axons, far away from neuronal cell bodies. Here, we report a physical and functional interaction between locally synthesized RPs and ribosomes in the axon. We show that axonal RP translation is regulated through a sequence motif, CUIC, that forms an RNA-loop structure in the region immediately upstream of the initiation codon. Using imaging and subcellular proteomics techniques, we show that RPs synthesized in axons join axonal ribosomes in a nucleolus-independent fashion. Inhibition of axonal CUIC-regulated RP translation decreases local translation activity and reduces axon branching in the developing brain, revealing the physiological relevance of axonal RP synthesis in vivo. These results suggest that axonal translation supplies cytoplasmic RPs to maintain/modify local ribosomal function far from the nucleolus in neurons.

Receptor-specific interactome as a hub for rapid cue-induced selective translation in axons.

Extrinsic cues trigger the local translation of specific mRNAs in growing axons via cell surface receptors. The coupling of ribosomes to receptors has been proposed as a mechanism linking signals to local translation but it is not known how broadly this mechanism operates, nor whether it can selectively regulate mRNA translation. We report that receptor-ribosome coupling is employed by multiple guidance cue receptors and this interaction is mRNA-dependent. We find that different receptors associate with distinct sets of mRNAs and RNA-binding proteins. Cue stimulation of growing Xenopus retinal ganglion cell axons induces rapid dissociation of ribosomes from receptors and the selective translation of receptor-specific mRNAs. Further, we show that receptor-ribosome dissociation and cue-induced selective translation are inhibited by co-exposure to translation-repressive cues, suggesting a novel mode of signal integration. Our findings reveal receptor-specific interactomes and suggest a generalizable model for cue-selective control of the local proteome.

Genetic control of cellular morphogenesis in Müller glia.

Cell shape is critical for the proper function of every cell in every tissue in the body. This is especially true for the highly morphologically diverse neural and glia cells of the central nervous system. The molecular processes by which these, or indeed any, cells gain their particular cell-specific morphology remain largely unexplored. To identify the genes involved in the morphogenesis of the principal glial cell type in the vertebrate retina, the Müller glia (MG), we used genomic and CRISPR based strategies in zebrafish (Danio rerio). We identified 41 genes involved in various aspects of MG cell morphogenesis and revealed a striking concordance between the sequential steps of anatomical feature addition and the expression of cohorts of functionally related genes that regulate these steps. We noted that the many of the genes preferentially expressed in zebrafish MG showed conservation in glia across species suggesting evolutionarily conserved glial developmental pathways.

Late Endosomes Act as mRNA Translation Platforms and Sustain Mitochondria in Axons.

Local translation regulates the axonal proteome, playing an important role in neuronal wiring and axon maintenance. How axonal mRNAs are localized to specific subcellular sites for translation, however, is not understood. Here we report that RNA granules associate with endosomes along the axons of retinal ganglion cells. RNA-bearing Rab7a late endosomes also associate with ribosomes, and real-time translation imaging reveals that they are sites of local protein synthesis. We show that RNA-bearing late endosomes often pause on mitochondria and that mRNAs encoding proteins for mitochondrial function are translated on Rab7a endosomes. Disruption of Rab7a function with Rab7a mutants, including those associated with Charcot-Marie-Tooth type 2B neuropathy, markedly decreases axonal protein synthesis, impairs mitochondrial function, and compromises axonal viability. Our findings thus reveal that late endosomes interact with RNA granules, translation machinery, and mitochondria and suggest that they serve as sites for regulating the supply of nascent pro-survival proteins in axons.

Disaggregation and Reaggregation of Zebrafish Retinal Cells for the Analysis of Neuronal Layering.

The reaggregation of dissociated cells to form organotypic structures provides an in vitro system for the analysis of the cellular interactions and molecular mechanisms involved in the formation of tissue architecture. The retina, an outgrowth of the forebrain, is a precisely layered neural tissue, yet the mechanisms underlying layer formation are largely unexplored. Here we describe the protocol to dissociate, re-aggregate, and culture zebrafish retinal cells from a transgenic, Spectrum of Fates, line where all main cell types are labelled with a combination of fluorescent proteins driven by fate-specific promoters. These cells re-aggregate and self-organize in just 48 h in minimal culture conditions. We also describe how the patterning in these aggregates can be analyzed using isocontour profiling to compare whether different conditions affect their self-organization.

Single-molecule analysis of endogenous ß-actin mRNA trafficking reveals a mechanism for compartmentalized mRNA localization in axons.

During embryonic nervous system assembly, mRNA localization is precisely regulated in growing axons, affording subcellular autonomy by allowing controlled protein expression in space and time. Different sets of mRNAs exhibit different localization patterns across the axon. However, little is known about how mRNAs move in axons or how these patterns are generated. Here, we couple molecular beacon technology with highly inclined and laminated optical sheet microscopy to image single molecules of identified endogenous mRNA in growing axons. By combining quantitative single-molecule imaging with biophysical motion models, we show that ß-actin mRNA travels mainly as single copies and exhibits different motion-type frequencies in different axonal subcompartments. We find that ß-actin mRNA density is fourfold enriched in the growth cone central domain compared with the axon shaft and that a modicum of directed transport is vital for delivery of mRNA to the axon tip. Through mathematical modeling we further demonstrate that directional differences in motor-driven mRNA transport speeds are sufficient to generate ß-actin mRNA enrichment at the growth cone. Our results provide insight into how mRNAs are trafficked in axons and a mechanism for generating different mRNA densities across axonal subcompartments.

Youth Risk Behavior Surveillance - United States, 2017.

PROBLEM: Health-risk behaviors contribute to the leading causes of morbidity and mortality among youth and adults in the United States. In addition, significant health disparities exist among demographic subgroups of youth defined by sex, race/ethnicity, and grade in school and between sexual minority and nonsexual minority youth. Population-based data on the most important health-related behaviors at the national, state, and local levels can be used to help monitor the effectiveness of public health interventions designed to protect and promote the health of youth at the national, state, and local levels. REPORTING PERIOD COVERED: September 2016-December 2017. DESCRIPTION OF THE SYSTEM: The Youth Risk Behavior Surveillance System (YRBSS) monitors six categories of priority health-related behaviors among youth and young adults: 1) behaviors that contribute to unintentional injuries and violence; 2) tobacco use; 3) alcohol and other drug use; 4) sexual behaviors related to unintended pregnancy and sexually transmitted infections (STIs), including human immunodeficiency virus (HIV) infection; 5) unhealthy dietary behaviors; and 6) physical inactivity. In addition, YRBSS monitors the prevalence of other health-related behaviors, obesity, and asthma. YRBSS includes a national school-based Youth Risk Behavior Survey (YRBS) conducted by CDC and state and large urban school district school-based YRBSs conducted by state and local education and health agencies. Starting with the 2015 YRBSS cycle, a question to ascertain sexual identity and a question to ascertain sex of sexual contacts were added to the national YRBS questionnaire and to the standard YRBS questionnaire used by the states and large urban school districts as a starting point for their questionnaires. This report summarizes results from the 2017 national YRBS for 121 health-related behaviors and for obesity, overweight, and asthma by demographic subgroups defined by sex, race/ethnicity, and grade in school and by sexual minority status; updates the numbers of sexual minority students nationwide; and describes overall trends in health-related behaviors during 1991-2017. This reports also summarizes results from 39 state and 21 large urban school district surveys with weighted data for the 2017 YRBSS cycle by sex and sexual minority status (where available). RESULTS: Results from the 2017 national YRBS indicated that many high school students are engaged in health-risk behaviors associated with the leading causes of death among persons aged 10-24 years in the United States. During the 30 days before the survey, 39.2% of high school students nationwide (among the 62.8% who drove a car or other vehicle during the 30 days before the survey) had texted or e-mailed while driving, 29.8% reported current alcohol use, and 19.8% reported current marijuana use. In addition, 14.0% of students had taken prescription pain medicine without a doctor's prescription or differently than how a doctor told them to use it one or more times during their life. During the 12 months before the survey, 19.0% had been bullied on school property and 7.4% had attempted suicide. Many high school students are engaged in sexual risk behaviors that relate to unintended pregnancies and STIs, including HIV infection. Nationwide, 39.5% of students had ever had sexual intercourse and 9.7% had had sexual intercourse with four or more persons during their life. Among currently sexually active students, 53.8% reported that either they or their partner had used a condom during their last sexual intercourse. Results from the 2017 national YRBS also indicated many high school students are engaged in behaviors associated with chronic diseases, such as cardiovascular disease, cancer, and diabetes. Nationwide, 8.8% of high school students had smoked cigarettes and 13.2% had used an electronic vapor product on at least 1 day during the 30 days before the survey. Forty-three percent played video or computer games or used a computer for 3 or more hours per day on an average school day for something that was not school work and 15.4% had not been physically active for a total of at least 60 minutes on at least 1 day during the 7 days before the survey. Further, 14.8% had obesity and 15.6% were overweight. The prevalence of most health-related behaviors varies by sex, race/ethnicity, and, particularly, sexual identity and sex of sexual contacts. Specifically, the prevalence of many health-risk behaviors is significantly higher among sexual minority students compared with nonsexual minority students. Nonetheless, analysis of long-term temporal trends indicates that the overall prevalence of most health-risk behaviors has moved in the desired direction. INTERPRETATION: Most high school students cope with the transition from childhood through adolescence to adulthood successfully and become healthy and productive adults. However, this report documents that some subgroups of students defined by sex, race/ethnicity, grade in school, and especially sexual minority status have a higher prevalence of many health-risk behaviors that might place them at risk for unnecessary or premature mortality, morbidity, and social problems (e.g., academic failure, poverty, and crime). PUBLIC HEALTH ACTION: YRBSS data are used widely to compare the prevalence of health-related behaviors among subpopulations of students; assess trends in health-related behaviors over time; monitor progress toward achieving 21 national health objectives; provide comparable state and large urban school district data; and take public health actions to decrease health-risk behaviors and improve health outcomes among youth. Using this and other reports based on scientifically sound data is important for raising awareness about the prevalence of health-related behaviors among students in grades 9-12, especially sexual minority students, among decision makers, the public, and a wide variety of agencies and organizations that work with youth. These agencies and organizations, including schools and youth-friendly health care providers, can help facilitate access to critically important education, health care, and high-impact, evidence-based interventions.

Axon-Axon Interactions Regulate Topographic Optic Tract Sorting via CYFIP2-Dependent WAVE Complex Function.

The axons of retinal ganglion cells (RGCs) are topographically sorted before they arrive at the optic tectum. This pre-target sorting, typical of axon tracts throughout the brain, is poorly understood. Here, we show that cytoplasmic FMR1-interacting proteins (CYFIPs) fulfill non-redundant functions in RGCs, with CYFIP1 mediating axon growth and CYFIP2 specifically involved in axon sorting. We find that CYFIP2 mediates homotypic and heterotypic contact-triggered fasciculation and repulsion responses between dorsal and ventral axons. CYFIP2 associates with transporting ribonucleoprotein particles in axons and regulates translation. Axon-axon contact stimulates CYFIP2 to move into growth cones where it joins the actin nucleating WAVE regulatory complex (WRC) in the periphery and regulates actin remodeling and filopodial dynamics. CYFIP2's function in axon sorting is mediated by its binding to the WRC but not its translational regulation. Together, these findings uncover CYFIP2 as a key regulatory link between axon-axon interactions, filopodial dynamics, and optic tract sorting.

RNA Docking and Local Translation Regulate Site-Specific Axon Remodeling In Vivo.

Nascent proteins can be positioned rapidly at precise subcellular locations by local protein synthesis (LPS) to facilitate localized growth responses. Axon arbor architecture, a major determinant of synaptic connectivity, is shaped by localized growth responses, but it is unknown whether LPS influences these responses in vivo. Using high-resolution live imaging, we examined the spatiotemporal dynamics of RNA and LPS in retinal axons during arborization in vivo. Endogenous RNA tracking reveals that RNA granules dock at sites of branch emergence and invade stabilized branches. Live translation reporter analysis reveals that de novo ß-actin hotspots colocalize with docked RNA granules at the bases and tips of new branches. Inhibition of axonal ß-actin mRNA translation disrupts arbor dynamics primarily by reducing new branch emergence and leads to impoverished terminal arbors. The results demonstrate a requirement for LPS in building arbor complexity and suggest a key role for pre-synaptic LPS in assembling neural circuits.

Mechanisms of Müller glial cell morphogenesis.

Müller Glia (MG), the radial glia cells of the retina, have spectacular morphologies subserving their enormous functional complexity. As early as 1892, the great neuroanatomist Santiago Ramon y Cajal studied the morphological development of MG, defining several steps in their morphogenesis [1,2]. However, the molecular cues controlling these developmental steps remain poorly understood. As MG have roles to play in every cellular and plexiform layer, this review discusses our current understanding on how MG morphology may be linked to their function, including the developmental mechanisms involved in MG patterning and morphogenesis. Uncovering the mechanisms governing glial morphogenesis, using transcriptomics and imaging, may provide shed new light on the pathophysiology and treatment of human neurological disorders.

Induction of Hypoxia in Living Frog and Zebrafish Embryos.

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h. To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1α) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2'-deoxyuridine (EdU) incorporation into the DNA. HIF-1α levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos. Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

In vivo expression of Nurr1/Nr4a2a in developing retinal amacrine subtypes in zebrafish Tg(nr4a2a:eGFP) transgenics.

The Nuclear receptor subfamily 4 group A member 2 (Nr4a2) is crucial for the formation or maintenance of dopaminergic neurons in the central nervous system including the retina, where dopaminergic amacrine cells contribute to visual function. Little is known about which cells express Nr4a2 at which developmental stage. Furthermore, whether Nr4a2 functions in combination with other genes is poorly understood. Thus, we generated a novel transgenic to visualize Nr4a2 expression in vivo during zebrafish retinogenesis. A 4.1 kb fragment of the nr4a2a promoter was used to drive green fluorescent protein expression in this Tg(nr4a2a:eGFP) line. In situ hybridization showed that transgene expression follows endogenous RNA expression at a cellular level. Temporal expression and lineages were quantified using in vivo time-lapse imaging in embryos. Nr4a2 expressing retinal subtypes were characterized immunohistochemically. Nr4a2a:eGFP labeled multiple neuron subtypes including 24.5% of all amacrine interneurons. Nr4a2a:eGFP labels all tyrosine hydroxylase labeled dopaminergic amacrine cells, and other nondopaminergic GABAergic amacrine populations. Nr4a2a:eGFP is confined to a specific progenitor lineage identified by sequential expression of the bhlh transcription factor Atonal7 (Atoh7) and Pancreas transcription factor 1a (Ptf1a), and labels postmitotic postmigratory amacrine cells. Thus, developmental Nr4a2a expression indicates a role during late differentiation of specific amacrine interneurons. Tg(nr4a2a:eGFP) is an early marker of distinct neurons including dopaminergic amacrine cells. It can be utilized to assess consequences of gene manipulations and understand whether Nr4a2 only carries out its role in the presence of specific coexpressed genes. This will allow Nr4a2 use to be refined for regenerative approaches.

Self-organising aggregates of zebrafish retinal cells for investigating mechanisms of neural lamination.

To investigate the cell-cell interactions necessary for the formation of retinal layers, we cultured dissociated zebrafish retinal progenitors in agarose microwells. Within these wells, the cells re-aggregated within hours, forming tight retinal organoids. Using a Spectrum of Fates zebrafish line, in which all different types of retinal neurons show distinct fluorescent spectra, we found that by 48 h in culture, the retinal organoids acquire a distinct spatial organisation, i.e. they became coarsely but clearly laminated. Retinal pigment epithelium cells were in the centre, photoreceptors and bipolar cells were next most central and amacrine cells and retinal ganglion cells were on the outside. Image analysis allowed us to derive quantitative measures of lamination, which we then used to find that Müller glia, but not RPE cells, are essential for this process.

A Novel Tool to Measure Extracellular Glutamate in the Zebrafish Nervous System In Vivo.

Glutamate is the major excitatory neurotransmitter in the brain. Its release and eventual recycling are key to rapid sustained neural activity. We have paired the gfap promoter region with the glutamate reporter molecule, iGluSnFR, to drive expression in glial cells throughout the nervous system. Tg(gfap:iGluSnFR) is expressed on the glial membrane of Müller glia cells in the retina, which rapidly respond to stimulation and the release of extracellular glutamate. As glial cells are associated with most, if not all, synapses, Tg(gfap:iGluSnFR) is a novel and exciting tool to measure neuronal activity and extracellular glutamate dynamics in many regions of the nervous system.

Hermes Regulates Axon Sorting in the Optic Tract by Post-Trancriptional Regulation of Neuropilin 1.

The establishment of precise topographic maps during neural development is facilitated by the presorting of axons in the pathway before they reach their targets. In the vertebrate visual system, such topography is seen clearly in the optic tract (OT) and in the optic radiations. However, the molecular mechanisms involved in pretarget axon sorting are poorly understood. Here, we show in zebrafish that the RNA-binding protein Hermes, which is expressed exclusively in retinal ganglion cells (RGCs), is involved in this process. Using a RiboTag approach, we show that Hermes acts as a negative translational regulator of specific mRNAs in RGCs. One of these targets is the guidance cue receptor Neuropilin 1 (Nrp1), which is sensitive to the repellent cue Semaphorin 3A (Sema3A). Hermes knock-down leads to topographic missorting in the OT through the upregulation of Nrp1. Restoring Nrp1 to appropriate levels in Hermes-depleted embryos rescues this effect and corrects the axon-sorting defect in the OT. Our data indicate that axon sorting relies on Hermes-regulated translation of Nrp1. SIGNIFICANCE STATEMENT: An important mechanism governing the formation of the mature neural map is pretarget axon sorting within the sensory tract; however, the molecular mechanisms involved in this process remain largely unknown. The work presented here reveals a novel function for the RNA-binding protein Hermes in regulating the topographic sorting of retinal ganglion cell (RGC) axons in the optic tract and tectum. We find that Hermes negatively controls the translation of the guidance cue receptor Neuropilin-1 in RGCs, with Hermes knock-down resulting in aberrant growth cone cue sensitivity and axonal topographic misprojections. We characterize a novel RNA-based mechanism by which axons restrict their translatome developmentally to achieve proper targeting.