Journal: Frontiers in Synaptic Neuroscience

Article Title: Patterned pre-sensory spontaneous activity drives the structural refinement of developing cochlear ribbon synapses

doi: 10.3389/fnsyn.2026.1730181

Figure Lengend Snippet: 2D-STED nanoscopy reveals a positive correlation between the sizes of ribbons and Ca v 1.3 clusters. (A) Representative examples of single optical sections of IHC synapses stained with antibodies against Ca v 1.3 (green; KO-verified) and CtBP2 (magenta) visualized using 2-color 2D-STED nanoscopy, while Homer1 (blue) – indicating the PSD context – was acquired in confocal mode. The individual image dimensions are 1 × 1 μm. (B) Presynaptic ribbon and Ca v 1.3 cluster sizes obtained from 2D-STED images display a moderate positive correlation as assessed by CtBP2 and Ca v 1.3 areas. The red line indicates a linear regression with the associated 95% confidence interval. N animals = 3, n ribbons = 99; r s = 0.35, *** p = 0.0004.

Article Snippet: For all conditions except Ca v 1.3 -KO, datasets were analyzed in Imaris x64 9.6.1, with 3D reconstructions of ribbon puncta generated using the surface algorithm with local background subtraction enabled.

Techniques: Staining

Journal: Frontiers in Synaptic Neuroscience

Article Title: Patterned pre-sensory spontaneous activity drives the structural refinement of developing cochlear ribbon synapses

doi: 10.3389/fnsyn.2026.1730181

Figure Lengend Snippet: Effects of acute pharmacological inhibition and genetics-based functional disruption of murine auditory ribbon synapses. (A) Representative temporal maximum intensity projections of GCaMP6 fluorescence in afferent SGN terminals over a 10 min timelapse interval in an organotypically-cultured organ of Corti of early postnatal Snap25-T2A-GCaMP6s-D mice (P5DIV1). Pharmacological block of presynaptic Ca v 1.x-mediated Ca 2+ influx by application of isradipine (Isra., 10 μM) abolished the spontaneous synaptic activity detected in the postsynaptic SGNs; panels display a representative time interval prior (A) and post (A’) isradipine application. Here, across the analysis time window, early Ca 2+ waves are represented in dark blue, late activity waves in yellow – the latter are completely absent upon isradipine treatment, thus illustrating complete block of synaptic transmission in this condition upon wash-in of the drug. Scale bar: 10 μm. (B) Normalized GCaMP6 fluorescence intensity plots of four different organ of Corti cultures treated with isradipine, prior and post application. (B’) Quantification of the peak frequency in SGN terminal GCaMP6 fluorescence, showing a dramatic reduction of activity bursts upon isradipine application. ( N animals = 4, n Corti = 4, imaged prior and post application). Paired Student’s t -test: ** p < 0.01. (C,C’) Representative maximum projections of confocal z -stacks of WT organ of Corti cultures treated with either (C) vehicle (DMSO) or (C’) 10 μM isradipine for 1 h at P5DIV1. IHCs were stained against Parvalbumin (blue), RibeyeA (green) and PSD95 (magenta). Scale bars: 5 μm. (D) Average ribbon counts in vehicle- and isradipine-treated IHCs at P5DIV1 ( N animals = 4, n Corti = 4 for both conditions) reveal no statistically significant differences between treatments. Two-way ANOVA with Holm-Šídák’s multiple comparisons correction: n.s. = no significant. (E,E’) Violin plots showing (E) unchanged integrated fluorescence intensity of presynaptic ribbons, but (E’) an increase in PSD integrated fluorescence intensity in isradipine-treated IHCs at P5DIV1 after 1 h of treatment. N animals = 4, n Corti = 4, n ribbons = 2,524, n PSD = 1,038 for vehicle- and N animals = 4, n Corti = 4, n ribbons = 2,713, n PSD = 1,076 for isradipine-treated conditions for both conditions. Median is indicated by solid lines inside the violin plots, while upper and lower quartiles are indicated by dashed lines. Mann–Whitney U -test: *** p < 0.001. (F,F’) Representative maximum projections of confocal z -stacks of (F) WT and (F’) Ca v 1.3 -KO organ of Corti cultures at P5DIV1. IHCs were stained against Parvalbumin (blue), RibeyeA (green) and PSD95 (magenta). Scale bars: 5 μm. (G) Quantification of average ribbon counts reveals a decrease in cytosolic ribbons in Ca v 1.3 -KO IHCs at P5DIV1 ( N animals = 3, n Corti = 6 for both genotypes). Two-way ANOVA with Holm-Šídák’s multiple comparisons correction: *** p < 0.001. (H,H’) Violin plots reveal (H) a decrease in ribbon integrated fluorescence intensity and (H’) an increase in PSD integrated fluorescence intensity in Ca v 1.3 -KO IHCs at P5DIV1. N animals = 3, n Corti = 6, n ribbons = 2,555, n PSD = 1,575 for each WT and N animals = 3, n Corti = 6, n ribbons = 2,555, n PSD = 1,466 for Ca v 1.3 -KOs. Median is indicated by solid lines inside the violin plots, while upper and lower quartiles are indicated by dashed lines. Mann–Whitney U -test: *** p < 0.001. (I,I’) Representative maximum projections of confocal z -stacks of (I) WT and (I’) Otof -KO organ of Corti cultures at P5DIV1. IHCs were stained against Parvalbumin (blue), RibeyeA (green) and PSD95 (magenta). Scale bars: 5 μm. (J) Quantification of synaptic as well as cytoplasmic ribbon counts reveals WT-like ribbon numbers in Otof -KO IHCs at P5DIV1 ( N animals = 3, n Corti = 6 for both genotypes). Two-way ANOVA with Holm-Šídák’s multiple comparisons correction. (K,K’) Violin plots showing (K) unchanged integrated fluorescence intensity in ribbons but (K’) an increase in PSD integrated fluorescence intensity in Otof -KO IHCs at P5DIV1. N animals = 3, n Corti = 6, n ribbons = 3,912, n PSD = 1,423 for each WT and N animals = 3, n Corti = 6, n ribbons = 3,105, n PSD = 1,064 Otof -KO. Median is indicated by solid lines inside the violin plots, while upper and lower quartiles are indicated by dashed lines. Mann–Whitney U -test: *** p < 0.001. Refer to for ribbon/PSD volume data.

Article Snippet: For all conditions except Ca v 1.3 -KO, datasets were analyzed in Imaris x64 9.6.1, with 3D reconstructions of ribbon puncta generated using the surface algorithm with local background subtraction enabled.

Techniques: Inhibition, Functional Assay, Disruption, Fluorescence, Cell Culture, Blocking Assay, Activity Assay, Transmission Assay, Staining, MANN-WHITNEY

Journal: Frontiers in Synaptic Neuroscience

Article Title: Patterned pre-sensory spontaneous activity drives the structural refinement of developing cochlear ribbon synapses

doi: 10.3389/fnsyn.2026.1730181

Figure Lengend Snippet: Summary of the observed structural changes of IHCs ribbon synapses following acute pharmacological, genetic, or optogenetic manipulation. Acute pharmacological inhibition of presynaptic Ca 2+ influx using the L-type calcium channel inhibitor isradipine, or functional disruption of exocytosis due to absence of Otoferlin, resulted in unchanged ribbon size but an increase in PSD size. Genetic ablation of Ca v 1.3 channels caused structural collapse of IHC ribbon size but also led to an increase in PSD size. Temporally-patterned optogenetic stimulation ( oSparse ) caused structural upscaling of both, ribbon and PSD size. In contrast, prolonged opening time of L-type calcium channels, induced by 1 h application of BayK led to a reduction in ribbon and PSD size. Notably, temporally-patterned optical stimulation in form of bursts ( oBurst ) similarly led to a reduction in ribbon size but failed to dramatically attenuate PSD size. Please note that we omitted possible postsynaptic Ca v 1. x channels from this schematic drawing due to current lack of experimental evidence for their presence in the PSD. PD, presynaptic density; SV, synaptic vesicle.

Article Snippet: For all conditions except Ca v 1.3 -KO, datasets were analyzed in Imaris x64 9.6.1, with 3D reconstructions of ribbon puncta generated using the surface algorithm with local background subtraction enabled.

Techniques: Inhibition, Functional Assay, Disruption

Journal: bioRxiv

Article Title: Comprehensive profiling of transcription factors for reprogramming human astrocytes to neuronal cells through endogenous CRISPR-based gene activation

doi: 10.1101/2025.10.11.681828

Figure Lengend Snippet: A. ORF overexpression of annotated INSM1, LHX6, and ZNF276 isoforms. RNA levels of neuron marker genes shown. *p < 0.05, ***p < 0.001 by global one-way ANOVA with Dunnett’s post hoc test comparing all groups to mCherry. B. Immunofluorescence staining of reprogrammed cells to assess MAP2, NeuN, and SLC17A7 expression 25 days after transduction. C. Number of spikes (neuronal firing events) in 5-minute multi-electrode array recording 32 days post-transduction. D. Flow analysis of TUBB3-2A-mCherry expression expression as proxy for early neuronal differentiation 5 days post-transduction of VP64 dSpCas9 VP iPSCs with gRNA. E. Summary of mouse gRNA sublibrary. F. Significance (P adj ) versus fold change in gRNA abundance between MAP2-high and MAP2-low populations in mouse CRISPRa screen. G. Top enriched biological processes for upregulated DEGs (L2FC >1, p adj <0.01 determined by DESeq2 vs mCherry, n=121 genes) from RNA-seq 10 days after INSM1 ORF overexpression. Statistical significance of term enrichment was determined using a two-tailed Fisher’s exact test followed by Benjamini–Hochberg correction.

Article Snippet: SLC17A7 screen: To screen for factors that cooperate with INSM1 to enhance glutamatergic subtype specification, cells were sorted based on abundance of SLC17A7 RNA using HCR-FlowFISH according to the method described in Reilly et al. 2021 with the following modifications: SLC17A7 probeset and buffers were ordered from Molecular Instruments ( https://www.molecularinstruments.com ), and SLC17A7 probeset was used at a final concentration of 8nM overnight.

Techniques: Over Expression, Marker, Immunofluorescence, Staining, Expressing, Transduction, RNA Sequencing, Two Tailed Test

Journal: bioRxiv

Article Title: Comprehensive profiling of transcription factors for reprogramming human astrocytes to neuronal cells through endogenous CRISPR-based gene activation

doi: 10.1101/2025.10.11.681828

Figure Lengend Snippet: A. Schematic of paired CRISPRa screens. B. Summary of TFpaired screening library. C. Scatter plot of z-score of gRNA abundance in the INSM1 MAP2 paired screen and the INSM1 SLC17A7 paired screen. D. Euler diagrams of differentially accessible peaks. Differential peaks (p adj <.01) for each sample were determined by DESeq2 vs. non-targeting gRNA. E. Top enriched biological processes for genes nearest top 1000 differentially accessible peaks by z-score for INSM1 (I), IKZF1 (IK), or peaks unique in only the combination of INSM1-IKZF1 (I+IK). Statistical significance of term enrichment was determined using a two-tailed Fisher’s exact test followed by Benjamini–Hochberg correction. F. Browser tracks of ATAC-seq (reads per kilobase per million mapped reads [RPKM]-normalized BigWig, bin size = 25bp. ‘Diff.peaks’ denotes peak significance between IKZF1-INSM1 and non-targeting using DESeq2. G. ANKS1B and KALRN peak accessibility and RNA expression indicate lack of significance after reprogramming with individual factors but significance after reprogramming with combination. H. Expression level ofANKS1B and KALRN in neural cell types in the Human Protein Atlas Single Cell Type data .

Article Snippet: SLC17A7 screen: To screen for factors that cooperate with INSM1 to enhance glutamatergic subtype specification, cells were sorted based on abundance of SLC17A7 RNA using HCR-FlowFISH according to the method described in Reilly et al. 2021 with the following modifications: SLC17A7 probeset and buffers were ordered from Molecular Instruments ( https://www.molecularinstruments.com ), and SLC17A7 probeset was used at a final concentration of 8nM overnight.

Techniques: Two Tailed Test, RNA Expression, Expressing