1. Neuroscience

Probing the role of synaptic adhesion molecule RTN4RL2 in setting up cochlear connectivity

  1. Nare Karagulyan
  2. Maja Überegger
  3. Yumeng Qi
  4. Norbert Babai
  5. Florian Hofer
  6. Lejo Johnson Chacko
  7. Fangfang Wang
  8. Maria Luque
  9. Rudolf Glueckert
  10. Anneliese Schrott-Fischer
  11. Yunfeng Hua  Is a corresponding author
  12. Tobias Moser  Is a corresponding author
  13. Christine Bandtlow  Is a corresponding author
  1. Institute for Auditory Neuroscience, University Medical Center Göttingen, Germany
  2. Auditory Neuroscience and Synaptic Nanophysiology Group, Max Planck Institute for Multidisciplinary Sciences, Germany
  3. Cluster of Excellence “Multiscale Bioimaging: From Molecular Machines to Networks of Excitable Cells”, Germany
  4. Institute of Neurobiochemistry, Biocenter, Medical University of Innsbruck, Austria
  5. Shanghai Institute of Precision Medicine, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, China
  6. Department of Otorhinolaryngology, Medical University of Innsbruck, Austria
  7. Department for Hearing, Speech and Voice Disorders, Medical University of Innsbruck, Austria
https://doi.org/10.7554/eLife.103481.3
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Cite this article
  1. Nare Karagulyan
  2. Maja Überegger
  3. Yumeng Qi
  4. Norbert Babai
  5. Florian Hofer
  6. Lejo Johnson Chacko
  7. Fangfang Wang
  8. Maria Luque
  9. Rudolf Glueckert
  10. Anneliese Schrott-Fischer
  11. Yunfeng Hua
  12. Tobias Moser
  13. Christine Bandtlow
(2025)
Probing the role of synaptic adhesion molecule RTN4RL2 in setting up cochlear connectivity
eLife 14:RP103481.
https://doi.org/10.7554/eLife.103481.3
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12 figures, 1 table and 1 additional file

Figures

Figure 1
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RTN4RL2 mRNA expression in inner hair cells (IHCs) and spiral ganglion neurons (SGNs) of the mouse cochlea.

(A) RTN4RL2 is an LRR protein and is anchored to the cell membrane via glycosylphosphatidylinositol (GPI). In the nervous system, RTN4RL2 has been implicated to interact with MAG, versican, brain-specific angiogenesis inhibitor (BAI) (Venkatesh et al., 2005; Bäumer et al., 2014; Wang et al., 2021). Exemplary images of RNAscope ISH for Rtn4rl2 mRNA (green) in the otic vesicles of E11.5 (B) and E12.5 (C) mice. Scale bar = 50 µm. The developing organ of Corti region is marked with an asterisk and the spiral ganglion is indicated with an arrow. Exemplary images of Rtn4rl2 mRNA expression in hair cells of E16.5 (D) and p1 (E) mice. Hair cells are visualized with anti-Myo7a stainings. Scale bars = 10 µm. (F) Representative images of RNAscope ISH for Rtn4rl2 mRNA (green dots) combined with immunostaining for neuron-specific marker βIII-tubulin (gray) in paraffin sections of p40 RTN4RL2 WT and RTN4RL2 KO cochleae. Scale bars = 20 µm.

Figure 2 with 3 supplements
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Pre- and postsynaptic changes at inner hair cells (IHCs) of RTN4RL2 KO mice.

Maximum intensity projections of representative confocal stacks of IHCs from apical cochlear region of 3-week-old RTN4RL2 WT (A) and RTN4RL2 KO (B) mice immunolabeled against Vglut3, Homer1, and Ctbp2. Scale bar = 5 µm. Images on the right-hand side are zoomed into the synaptic regions. Scale bar = 2 µm. Some of the putative ‘orphan’ postsynaptic densities (PSDs) are marked with the white arrowheads. (C) Number of Ctbp2-positive puncta is not changed in IHCs of RTN4RL2 KO mice (RTN4RL2 WT: 9.9 ± 0.42, SD = 1.62, n = 15, N = 2 vs RTN4RL2 KO: 11.4 ± 0.5, SD = 2.25, n = 20, N = 3; p = 0.06, Mann–Whitney–Wilcoxon test). (D) Ribbon volumes are enlarged in RTN4RL2 KO IHCs (RTN4RL2 WT: 0.16 ± 0.007 µm3, SD = 0.09 µm3, n = 165, N = 2 vs RTN4RL2 KO: 0.21 ± 0.005 µm3, SD = 0.09 µm3, n = 259, N = 3; p < 0.001, Mann–Whitney–Wilcoxon test). (E) Homer1 patches which are juxtaposing presynaptic ribbons show decreased volumes in RTN4RL2 KO IHCs (RTN4RL2 WT: 0.36 ± 0.01 µm3, SD = 0.16 µm3, n = 160, N = 2 vs RTN4RL2 KO: 0.26 ± 0.01 µm3, SD = 0.19 µm3, n = 249, N = 3; p < 0.001, Mann–Whitney–Wilcoxon test). (F) Percentage of Ctbp2 puncta juxtaposing Homer1 is slightly decreased in RTN4RL2 KO mice (RTN4RL2 WT: 96.7 ± 1.45%, SD = 5.44 %, n = 14, N = 2 vs RTN4RL2 KO: 89.5 ± 2.05%, SD = 9.18 %, n = 20, N = 3; p = 0.03, Mann–Whitney–Wilcoxon test). Data is presented as mean ± SEM. Box–whisker plots show the median, 25/75 percentiles (box), and 10/90 percentiles (whiskers). Individual data points are overlaid. Significances are reported as *p < 0.05 and ***p < 0.001.

Figure 2—source data 1

Numerical data of Figure 2C–F.

https://cdn.elifesciences.org/articles/103481/elife-103481-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
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Cochlear cell densities are not changed in RTN4RL2 KO mice.

(A) Mid/basal-modiolar sections labeled for βIII-tubulin (green, neurons) from p40 RTN4RL2 WT and RTN4RL2 KO mice. Exemplary section for RTN4RL2 KO is the zoomed-out image presented in Figure 1C. (B) Quantitative analysis shows that the density of spiral ganglion neuron (SGNs/103µm2) cell bodies is similar between RTN4RL2 KO and control cochleae. Data is presented as mean ± SD; N = 7 per group. Scale bar = 10 µm. Inner hair cell (IHC) (C) and OHC (D) densities are not affected in P15, 1 month, and 2 months old RTN4RL2 KO mice.

Figure 2—figure supplement 2
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Intact number but enlarged size of the ribbons in RTN4RL2 KO inner hair cells (IHCs).

(A) Maximum intensity projections of representative IHCs from apical, mid, and basal regions of RTN4RL2 WT (left) and RTN4RL2 KO (right) cochleae of p21–30 mice. Synapses are visualized by staining against Ctbp2/Ribeye (ribbons) and Homer1 (postsynaptic densities, PSDs). Scale bar = 5 µm. (B) The number of the ribbons is not affected along the tonotopic axis in RTN4RL2 KO cochleae. (C) The size of the ribbons is increased in IHCs of both apical and middle turns in RTN4RL2 KO cochleae (p < 0.001, Mann–Whitney–Wilcoxon test). N = 6 animals/genotype. Box–whisker plots show the median, 25/75 percentiles (box), and the range (whiskers). Individual data points are overlaid. Significances are reported as ***p < 0.001.

Figure 2—figure supplement 3
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Efferent innervation pattern in RTN4RL2 KO cochleae.

(A) Maximum intensity projections of confocal stacks of outer and inner hair cell (IHC) regions at the apical region of the cochlea from p21–30 RTN4RL2 WT (left) and RTN4RL2 KO (right) mice. Spiral ganglion neuron (SGN) fibers/terminals and efferent terminals are visualized staining for Na+/K+ ATPase and vesicular acetylcholine transporter (VAChT), respectively. Scale bar = 10 µm (B). Same as (A) but zoomed into the IHC region. Scale bar = 5 μm. (C) Maximum intensity projections of confocal stacks of apical IHC regions from p21 RTN4RL2 WT (left) and RTN4RL2 KO (right) mice. Presynaptic efferent terminals were stained with an anti-synapsin1/2 antibody. Scale bar = 5 µm.

Figure 3 with 1 supplement
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Reduced GluA2/3 signal juxtaposing presynaptic ribbons in inner hair cells (IHCs) of RTN4RL2 KO mice.

(A) Maximum intensity projections of representative IHC regions from 1- to 1.5-month-old RTN4RL2 WT (top) and RTN4RL2 KO (bottom) mouse cochleae immunolabeled against Ctbp2/Ribeye (ribbons) and GluA2/3 (AMPA receptors of postsynaptic density [PSD]). Scale bar = 5 µm. The zoom-in regions marked with the white rectangles are presented on the right-hand side. Scale bar = 2 µm. (B) The number of the GluA2/3-positive puncta is drastically reduced in RTN4RL2 KO mice despite the maintained number of presynaptic ribbons (p < 0.001, Mann–Whitney–Wilcoxon test). (C) Disrupted colocalization of Ctbp2/Ribeye and GluA2 immunofluorescence puncta at IHCs of RTN4RL2 KO mice (p < 0.001, Mann–Whitney–Wilcoxon test). N = 6 animals/genotype. (D) Representative images of RNAscope ISH from p4 mice show maintained expression of Gria2 (red dots) in the spiral ganglion neuron (SGN) somata of RTN4RL2 KO mice. Scale bar = 20 µm. Significances are reported as ***p < 0.001.

Figure 3—figure supplement 1
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No apparent GluA4 signal juxtaposing presynaptic ribbons at inner hair cell (IHC) synapses of RTN4RL2 KO mice.

Maximum intensity projections of a few imaging planes from apical IHC synaptic regions of 14-week-old N = 3 RTN4RL2 WT (A) and N = 3 RTN4RL2 KO (B) mice immunostained against GluA4, GluA2, and RibeyeA. Scale bars = 2 µm. (C) Fluorescence intensities of GluA4 and GluA2 channels were analyzed as the sum of all pixel intensities in approximately 1 × 1 × 1 μm3 volumes around the center of mass of RibeyeA immunofluorescent puncta (red boxes, top image). Background GluA4 and GluA2 fluorescence signals were calculated in the same way but outside the ribbons, at random spots inside IHCs (pink boxes, bottom image). Images show the maximum intensity projection of the IHC region in an RTN4RL2 KO mouse. (D) Fluorescence intensities of GluA4 (top) and GluA2 (bottom) channels at ribbons of RTN4RL2 KO mice do not show a significant increase compared to the background (RTN4RL2 KO random) but are significantly decreased compared to RTN4RL2 WT (Mann–Whitney–Wilcoxon test followed by Bonferroni multiple-comparison correction). Box–whisker plots show the median, 25/75 percentiles (box), and 10/90 percentiles (whiskers). Individual data points are overlaid. Significances are reported as ***p < 0.001 and ****p < 0.0001.

Figure 3—figure supplement 1—source data 1

Numerical data of Figure 3—figure supplement 1D.

https://cdn.elifesciences.org/articles/103481/elife-103481-fig3-figsupp1-data1-v1.xlsx
Figure 4 with 1 supplement
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Additional non-synaptically engaged spiral ganglion neuron (SGN) neurites in the cochlea of RTN4RL2 KO mice.

(A) Workflow of serial block-face scanning electron microscopy (SBEM) imaging at the mouse apical cochlear region. (B) Example images of neurites beneath inner hair cells (IHCs) from the RTN4RL2 WT (left) and RTN4RL2 KO (right) mice. Synaptic ribbons are indicated with red arrows. The regions of interest were magnified from single sections of SBEM datasets (insets). Scale bar = 2 µm. (C) 3D rendering of afferent fiber reconstruction with ribbons (red), showing both synaptic (green, type I SGN with ribbon) and non-synaptic (gray, type I SGN without ribbon) populations in the RTN4RL2 KO mouse. Scale bar = 10 µm. Inset: EM images show the SGN segments on which myelin sheaths start to form. Scale bar 2 = μm. (D) Display of classified radial fibers in the RTN4RL2 WT (right) and RTN4RL2 KO (left and bottom) animals. All fibers were traced from the habenula perforata (cycles) before classification to avoid bias to terminal types. Scale bar = 10 μm. (E) Percentage of radial fibers with ribbon per bundle (RTN4RL2 WT: 91.41 ± 8.35%, n = 3 bundles, N = 1 vs RTN4RL2 KO: 74.40 ± 14.58%, n = 6 bundles, N = 2; p = 0.032, unpaired t-test). (F) Percentage of unbranched radial fibers per bundle (RTN4RL2 WT: 94.19 ± 5.04%, n = 3 bundles, N = 1 vs RTN4RL2 KO: 96.98 ± 3.49%, n = 6 bundles, N = 2; p = 0.226, unpaired t-test). Significances are reported as *p < 0.05.

Figure 4—source data 1

Numerical data of Figure 4E, F.

https://cdn.elifesciences.org/articles/103481/elife-103481-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
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Quantification of ribbon number and volume in serial block-face scanning electron microscopy (SBEM) reconstructions.

(A) The number of ribbons is not changed in RTN4RL2 KO inner hair cells (IHCs) (RTN4RL2 KO: 10.3 ± 0.61, SD = 2.1, n = 12, N = 2 vs RTN4RL2 WT: 10.8 ± 0.83, SD = 2.04, n = 6, N = 1; p = 0.54, Mann–Whitney–Wilcoxon test). (B) Ribbon volumes tend to be larger in RTN4RL2 KO IHCs without reaching statistical significance (RTN4RL2 KO: 0.024 ± 0.001 µm3, SD = 0.01 µm3, n = 125 ribbons in 12 IHCs, N = 2 vs RTN4RL2 WT: 0.021 ± 0.001 µm3, SD = 0.008 µm3, n = 65 ribbons in 6 IHCs, N = 1; p = 0.06, Mann–Whitney–Wilcoxon test). Box–whisker plots show the median, 25/75 percentiles (box), and the range (whiskers). Individual data points are overlaid.

Figure 4—figure supplement 1—source data 1

Numerical data of Figure 4—figure supplement 1A, B.

https://cdn.elifesciences.org/articles/103481/elife-103481-fig4-figsupp1-data1-v1.xlsx
Figure 5
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Shifted operation range of Ca2+ channels but intact exocytosis in inner hair cells (IHCs) of RTN4RL2 KO mice.

(A) Representative current traces from IHCs of RTN4RL2 WT (top, black) and RTN4RL2 WT (bottom, red) evoked by step depolarizations. (B) Average Ca2+ current–voltage relationships (IV curves) in RTN4RL2 WT and RTN4RL2 KO IHCs. (C) Maximal Ca2+ current amplitude is not changed in RTN4RL2 KO IHCs (RTN4RL2 WT: 210 ± 10.9 pA, SD = 40.9 pA, n = 14, N = 5 vs RTN4RL2 KO: –200 ± 8.19 pA, SD = 40.9 pA, n = 25, N = 8; p = 0.47, Student’s t-test). (D) Fractional activation curves of Ca2+ channels calculated from the IV curves show depolarized shift in channel activation in RTN4RL2 KO IHCs. (E) Voltage of half maximal activation obtained from Boltzmann fit of the curves from (D) is more positive in RTN4RL2 KO IHCs (RTN4RL2 WT: –29.4 ± 0.66 mV, SD = 2.48 mV, n = 14, N = 5 vs RTN4RL2 KO: –26.6 ± 0.59 mV, SD = 2.94 mV, n = 25, N = 8; p = 0.004, Student’s t-test). (F) Voltage sensitivity (k) is not changed in RTN4RL2 KO IHCs (RTN4RL2 WT: 6.92 ± 0.12 mV, SD = 0.46 mV, n = 14, N = 5 vs RTN4RL2 KO: 6.98 ± 0.1 mV, SD = 0.51 mV, n = 25, N = 8; p = 0.93, Mann–Whitney–Wilcoxon test). (G) Average current traces evoked by 50 ms depolarization to –17 mV (top row) and resulting capacitance response (bottom row) from RTN4RL2 WT (left, black) and RTN4RL2 KO (right, red) IHCs. Shaded areas represent ± SEM. (H) Exocytic capacitance change (△Cm, top) and corresponding Ca2+ charge (QCa2+, bottom) evoked by depolarizations (to –17 mV) of various durations (2, 5, 10, 20, 50, and 100 ms). Box–whisker plots show the median, 25/75 percentiles (box), and 10/90 percentiles (whiskers). Individual data points are overlaid. Significances are reported as **p < 0.01.

Figure 5—source data 1

Numerical data of Figure 5B–F, H.

https://cdn.elifesciences.org/articles/103481/elife-103481-fig5-data1-v1.xlsx
Figure 6
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Depolarized shift of Ca2+ channel activation at single active zones (AZs) but intact presynaptic organization in RTN4RL2 KO inner hair cells (IHCs).

(A) Voltage ramp stimulation protocols (top), evoked whole-cell currents (middle), and the presynaptic hotspots of Fluo4-FF fluorescence (bottom) of a representative IHC recording. Black and gray colors represent the two stimulations, one being 5 ms shifted over the other. Images on the right show single imaging planes of representative RTN4RL2 WT (left) and RTN4RL2 KO (right) IHCs filled with TAMRA-conjugated Ctbp2 binding peptide (Ctbp2 bp) and Fluo4-FF Ca2+ dye. Ca2+ hotspots are visualized by subtracting the average of baseline planes from the average of 5 planes during stimulation. Scale bar = 2 µm. (B) Average fluorescence–voltage relationships of Ca2+ influx at single AZ from RTN4RL2 WT and RTN4RL2 KO IHCs show no difference in the maximal Ca2+ amplitude (Bi; RTN4RL2 WT: 1.6 ± 0.17, SD = 1.2, n = 50 AZs vs RTN4RL2 KO: 1.7 ± 0.13 pA, SD = 1.09, n = 69 AZs; p = 0.24, Mann–Whitney–Wilcoxon test). Shaded areas represent ± SEM. (C) Average fractional activation curves of Ca2+ channels at single AZs show intact voltage sensitivity (Ci; RTN4RL2 WT: 5.79 ± 0.32 mV, SD = 2.04 mV, n = 42 AZs vs RTN4RL2 KO: 6.25 ± 0.22 mV, SD = 1.7 mV, n = 59 AZs; p = 0.06, Mann–Whitney–Wilcoxon test) but depolarized shift of Vhalf (Cii; RTN4RL2 WT: –30 ± 1 mV, SD = 6.5 mV, n = 42 AZs vs RTN4RL2 KO: –25.5 ± 0.98 mV, SD = 7.49 mV, n = 59 AZs; p = 0.002, Student’s t-test) in RTN4RL2 KO IHCs. Shaded areas represent ± SEM. (D) Representative immunolabelings of presynaptic proteins show no apparent mislocalization in RTN4RL2 KO IHCs. Scale bar = 5 µm. Box–whisker plots show the median, 25/75 percentiles (box), and 10/90 percentiles (whiskers). Individual data points are overlaid. Significances are reported as **p < 0.01.

Figure 6—source data 1

Numerical data of Figure 6B, C, Bi–Cii.

https://cdn.elifesciences.org/articles/103481/elife-103481-fig6-data1-v1.xlsx
Figure 7
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Elevated acoustic thresholds in RTN4RL2 KO mice.

Auditory brainstem response (ABR) thresholds were measured in response to 4, 8, 16, and 32 kHz tone bursts and click stimuli. ABR thresholds of individual animals are shown in open circles on top of the mean ± SEM. Statistical significances are reported as *p < 0.05, ***p < 0.001, Kruskal–Wallis followed by Dunn’s multiple comparison test.

Figure 7—source data 1

Numerical data of Figure 7.

https://cdn.elifesciences.org/articles/103481/elife-103481-fig7-data1-v1.xlsx
Figure 8
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Schematic illustration of the key structural and functional changes in the auditory periphery of RTN4RL2 KO mice.

RTN4RL2 KO mice display enlarged synaptic ribbons and depolarized shift in the activation of presynaptic Ca2+ channels in inner hair cells (IHCs), as well as reduced size of postsynaptic densities (PSDs) juxtaposing presynaptic ribbons. RTN4RL2 deficiency further leads to a decrease in GluA2–4 AMPA receptor subunits at PSDs and results in a subset of type I spiral ganglion neuron (SGN) neurites that reach the inner spiral bundle but do not engage the IHCs.

Author response image 1
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Author response image 2
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Calretinin intensity distribution in spiral ganglion of RTN4RL2+/+ and RTN4RL2-/- mice.

(A) Mid-modiolar cochlear cryosections from RTN4RL2+/+ (top) and RTN4RL2-/- (bottom) mice immunolabeled against Parvalbumin (PV) and Calretinin (CR). Scale bar = 20 mm. (B) Distribution of CR intensity in PV positive cells (N = 3 for each genotype). (C) Distribution of the ratio of CR and PV intensities (N = 3 for each genotype).

Author response image 3
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The modiolar-pillar gradient of ribbon size is preserved in RTN4RL2-/- IHCs.

(A) Maximum intensity projections of approximately 2 IHCs stained against Vglut3 and Ctbp2 from 14-week-old RTN4RL2+/+ (left) and RTN4RL2-/- (right) mice. Scale bar = 5 mm. (B) Synaptic ribbons on the modiolar side show higher fluorescence intensity than the ones on the pillar side of mid-cochlear IHCs in both RTN4RL2+/+ (left, N=2) RTN4RL2-/- (right, N=2) mice. (C) Average fluorescence intensity of modiolar ribbons per IHC is higher than the average fluorescence intensity of pillar ribbons (paired t-test, p < 0.001).

Author response image 4
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Attempts to localize “orphan” Homer1 patches on type I SGN neurites.

(A) Single exemplary imaging planes of apical IHC region from RTN4RL2+/+ (left) and RTN4RL2-/- (right) mice immunolabeled against NF200, Vglut3 and Homer1. White arrows show putative “orphan” Homer1 puncta on NF200 positive neurites. Scale bar = 5 mm. (B) Maximum intensity projections of representative confocal stacks of IHCs from RTN4RL2-/- mice immunolabeled against Calretinin and Homer1. Scale bars = 5 mm. White arrows show possible “orphan” Homer1 puncta on Calretinin positive boutons.

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Genetic reagent (Mus musculus)RTN4RL2 KOWörter et al., 2009
Antibodyanti-Homer1
(rabbit polyclonal)		Synaptic SystemsCat# 1600021:500
Antibodyanti-Ctbp2
(mouse monoclonal)		BD BiosciencesCat# 6120441:200
KO validation; https://doi.org/10.7554/eLife.30241 https://doi.org/10.7554/eLife.29275
Antibodyanti-CaV1.3
(rabbit polyclonal)		Alomone LabsCat# ACC-0051:100
KO validation; https://doi.org/10.1523/JNEUROSCI.3411-05.2005
Antibodyanti-Bassoon
(mouse monoclonal)		AbcamCat# ab82981:300
Antibodyanti-GluA2
(mouse monoclonal)		MilliporeCat# MAB3971:200
validated for use in ELISA, IC, IH, IP, RIA, and WB, refer to manufacturer’s datasheet
Antibodyanti-GluA2/3
(rabbit polyclonal)		ChemiconCat# AB15061:200
validated for use in IH(P), IC, IH, IP, and WB, refer to manufacturer’s datasheet
Antibodyanti-GluA4
(rabbit polyclonal)		MilliporeCat# AB15081:200
KO validation; https://doi.org/10.1126/sciadv.aax5936
Antibodyanti-Myosin7a
(rabbit polyclonal)		Abcam/Proteus BioSciencesCat# ab34811:800
Antibodyanti-RibeyeA
(guinea pig polyclonal)		Synaptic SystemsCat# 1921041:500
Antibodyanti-Synapsin 1/2
(guinea pig polyclonal)		Synaptic SystemsCat# 1060041:500
KO validation; refer to manufacturer’s datasheet
Antibodyanti-Vglut3
(guinea pig polyclonal)		Synaptic SystemsCat# 1352041:500
KO validation;
https://doi.org/10.3389/fncel.2017.00140
Antibodyanti-Parvalbumin
(chicken polyclonal)		Synaptic SystemsCat# 1950061:200
Antibodyanti-Calretinin
(chicken polyclonal)		Synaptic SystemsCat# 2141061:200
Antibodyanti-VaChT
(guinea pig polyclonal)		Synaptic SystemsCat# 1391051:1000
Antibodyanti-ATP1A3
(mouse monoclonal)		InvitrogenCat# MA3-9151:300
AntibodyβIII-tubulin (mouse monoclonal)BiolegendCat# 8012021:1000
AntibodyAlexa Fluor 488 conjugated anti-guinea pig
(goat polyclonal)		Thermo Fisher ScientificA110731:200
AntibodyAlexa Fluor 488 conjugated anti-rabbit
(goat polyclonal)		Thermo Fisher ScientificA110081:200
AntibodyAlexa Fluor 568 conjugated anti-chicken (goat polyclonal)Abcamab1757111:200
AntibodyAlexa Fluor 633 conjugated anti-guinea pig
(goat polyclonal)		Thermo Fisher ScientificA211051:200
AntibodySTAR 580 conjugated anti-mouse
(goat polyclonal)		AbberiorST580P-1001-500UG1:200
AntibodySTAR 635 conjugated anti-rabbit
(goat polyclonal)		AbberiorST635P-1002-500UG1:200
Commercial assay, kitRNAscopeAdvanced Cell Diagnostics
Sequence-based reagentGria2 probe (mouse)
RNAscope		Advanced Cell Diagnostics,Cat# 865091
Sequence-based reagentRtn4rl2 probe (mouse)
RNAscope		Advanced Cell Diagnostics,Cat# 450761
Software, algorithmwebKnossosBoergens et al., 2017https://webknossos.org
Software, algorithmAmiraThermo Scientific, UShttps://www.thermofisher.com/amira
Software, algorithmMATLABMathWorkshttps://www.mathworks.com/products/matlab.html
Software, algorithmIGOR ProWaveMetricsVersion 6.3, https://www.wavemetrics.com
Software, algorithmPatchers Power Toolshttp://www3.mpibpc.mpg.de/groups/neher/index.php?page=software
Software, algorithmImarisOxford Instruments (Bitplane)Version 9.6 https://imaris.oxinst.com
Software, algorithmFiji (ImageJ)Schindelin et al., 2012
https://doi.org/10.1038/nmeth.2019
https://fiji.sc/
Software, algorithmPythonPython Software FoundationVersion 3.8.17 https://www.python.org
Software, algorithmGraphPad PrismGraphPad Software Inchttps://www.graphpad.com/scientific-software/prism/

Software, algorithmInkscapeThe Inkscape ProjectVersion 1.3.2 https://inkscape.org/

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  1. Nare Karagulyan
  2. Maja Überegger
  3. Yumeng Qi
  4. Norbert Babai
  5. Florian Hofer
  6. Lejo Johnson Chacko
  7. Fangfang Wang
  8. Maria Luque
  9. Rudolf Glueckert
  10. Anneliese Schrott-Fischer
  11. Yunfeng Hua
  12. Tobias Moser
  13. Christine Bandtlow
(2025)
Probing the role of synaptic adhesion molecule RTN4RL2 in setting up cochlear connectivity
eLife 14:RP103481.
https://doi.org/10.7554/eLife.103481.3