Experience-dependent flexibility in a molecularly diverse central-to-peripheral auditory feedback system
- Department of Neurobiology, Harvard Medical School, United States
- Section on Neuronal Circuitry, National Institute on Deafness and Other Communication Disorders, United States
Figures
Figure 1 with 2 supplements
Identification of OCNs by single-nucleus sequencing of brainstem neurons.
(A, B) Schematic of medial (MOC) and lateral (LOC) olivocochlear neuron (OCN) subtypes housed in the brainstem (A) and their projections into the cochlea, shown in cross-section (A) and top-down (B).…
Figure 1—figure supplement 1
Quality-control metrics for single-nucleus sequencing data.
(A) UMAP plot showing contributions from each age collected. Each neuronal cluster includes cells from all three timepoints, indicating that the clusters are not being driven by variability across …
Figure 1—figure supplement 2
OCNs and FMNs are transcriptionally distinct.
(A) Etv1 is a transcription factor selectively expressed in a subset of FMNs (VII). It is primarily expressed in only one motor neuron cluster. (B) Epha7 was previously identified as a marker for a …
Figure 2 with 1 supplement
MOCs and LOCs exhibit distinct gene-expression profiles.
(A) Heatmap summarizing expression of the 10 genes with the highest average log-fold change for LOCs and MOCs. For visualization purposes, scaled expression levels were capped at –2.5 and 2.5. (B–G) …
Figure 2—figure supplement 1
OCN markers across postnatal development.
(A) Selected genes with significantly different expression between MOCs and LOCs across postnatal development. (p<0.05, Wilcoxon rank-sum test, Bonferroni post-hoc correction). For visualization …
Figure 3 with 1 supplement
LOC neurons include a peptide-enriched subset.
(A–C) UMAPs summarizing sub-clustering results of 590 OCNs from 32 ChatCre; Rosa26Sun1-GFP animals (P26–P28). OCNs sub-cluster into five different groups (A). Feature plots denote normalized …
Figure 3—figure supplement 1
Additional metrics for OCN transcriptional subtypes.
(A–C) The number of genes detected (A), number of UMI (B), and percent of mitochondrial genes (C) is similar within MOC and LOC subtypes, indicating that differences between clusters are not …
Figure 4 with 1 supplement
Sparse labeling of OCNs reveals MOC connectivity in the cochlea.
(A–A”) Low doses of tamoxifen in RetCreER; Igs7GFP animals sparsely labels OCNs, enabling visualization of individual MOC axons (white) as they extend along and through the row of IHCs (CALB2, …
Figure 4—video 1
Terminal MOC axon morphology with putative synapses in the ISB and OHC region.
The full confocal z-stack volume of terminal MOC axons is shown, with CALB2 (blue) labeling IHCs and SGN peripheral fibers, the MOC axons labeled by RetCreER; Igs7GFP (white), and Syp labeling …
Figure 5 with 2 supplements
Anatomical heterogeneity in LOCs does not correlate with peptidergic identity.
(A) Schematic of the IHC region of the Organ of Corti. Peripheral fibers of Type I SGN subtypes (blue) innervate the IHCs in a low-to-high CALB2 expression gradient along the modiolar-pillar axis. (B…
Figure 5—figure supplement 1
Additional morphological quantification of OCN terminal axons.
(A, B) Ret is broadly expressed in OCNs at least as early as P1. (A) UMAP plot denoting Ret expression. Ret transcripts are detected in multiple motor neuron clusters, including OCNs. For …
Figure 5—video 1
Terminal LOC axon morphology with complex innervation patterns.
The full confocal z-stack volume is shown first, with CALB2 (blue) labeling IHCs and SGN peripheral fibers and the single LOC axon labeled by RetCreER; Igs7GFP (white), NPY (yellow), and Syp …
Figure 6
Single-cell electrophysiological measures do not correlate with GFP intensity in Calca-GFP+ LOCs.
(A–A’) Confocal Z-stack of an unfixed brain slice from a Calca-GFP mouse showing native GFP fluorescence in greyscale. Dashed line indicates boundaries of the LSO. (A’) Zoom of recording region …
Figure 7 with 1 supplement
Neuropeptide expression in LOCs emerges over postnatal development and is affected by hearing.
(A–D) Log-normalized counts of Calca (A), Calcb (B), Ucn (C), and Npy (D) expression across postnatal development as determined by single-nucleus sequencing. Mean ± SEM; Kruskal-Wallis with Dunn’s …
Figure 7—figure supplement 1
Quantification of additional neurotransmitters in TMC1/2dKO animals.
(A) Example of the cell segmentation and LSO normalization procedure for quantifying expression of neurotransmitters in LOCs. White line indicates the length of the LSO used for distance …
Figure 8
Neuropeptide expression in LOCs changes with sound exposure.
(A) Representative images of ChAT, TH, Ucn, NPY, and CGRP immunolabeling in unexposed (top) and sound-exposed (bottom) animals 9 days after a 2-hr exposure of 110 dB, 8–16 kHz noise. N=4 animals of …
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, C57Bl/6J (Mus musculus) | C57BL/6J | The Jackson Laboratory | RRID:IMSR_JAX:000664 | |
| Genetic reagent (Mus musculus) | Calca-GFP | Gensat | RRID:MMRRC_011187-UCD | Full name: Tg(Calca-EGFP)FG104Gsat/Mmucd |
| Genetic reagent (Mus musculus) | Rosa26tdTomato | The Jackson Laboratory | RRID:IMSR_JAX:007914 | Full name: B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J |
| Genetic reagent (Mus musculus) | Igs7GFP | The Jackson Laboratory | RRID:IMSR_JAX:030220 | Full name: B6.Cg-Igs7tm140.1(tetO-EGFP,CAG-tTA2)Hze/J |
| Genetic reagent (Mus musculus) | Rosa26Sun1-GFP | The Jackson Laboratory | RRID:IMSR_JAX:021039 | Full name: B6;129-Gt(ROSA)26Sortm5(CAG-Sun1/sfGFP)Nat/J |
| Genetic reagent (Mus musculus) | ChatCre | The Jackson Laboratory | RRID:IMSR_JAX:006410 | Full name: B6;129S6-Chattm2(cre)Lowl/J |
| Genetic reagent (Mus musculus) | ChatCreΔNeo | The Jackson Laboratory | RRID:IMSR_JAX:031661 | Full name: B6.129S-Chattm1(cre)Lowl/MwarJ |
| Genetic reagent (Mus musculus) | NpyFlpO | The Jackson Laboratory | RRID:IMSR_JAX:030211 | Full name: B6.Cg-Npytm1.1(flpo)Hze/J |
| Genetic reagent (Mus musculus) | RetCreER | David Ginty (Harvard); Luo et al., 2009 | MGI:4437245 | Full name: Rettm2(cre/ERT2)Ddg |
| Genetic reagent (Mus musculus) | Npy-GFP | The Jackson Laboratory | RRID:IMSR_JAX:006417 | Full name: B6.FVB-Tg(Npy-hrGFP)1Lowl/J |
| Genetic reagent (Mus musculus) | Tmc1KO | David Corey (Harvard); Kawashima et al., 2011 | MGI:5427097 | Full name: Tmc1tm1.1Ajg |
| Genetic reagent (Mus musculus) | Tmc2KO | David Corey (Harvard); Kawashima et al., 2011 | MGI:5427099 | Full name: Tmc2tm1.1Ajg |
| Genetic reagent (Mus musculus) | Rosa26FLTG | The Jackson Laboratory | RRID:IMSR_JAX:026932 | Full name: B6.Cg-Gt(ROSA)26Sortm1.3(CAG-tdTomato,-EGFP)Pjen/J |
| Antibody | Goat polyclonal Anti-Calb2 antiserum | Swant | Cat#CG1; RRID: AB_10000342 | (1:1,000) |
| Antibody | Goat polyclonal Anti-ChAT | Millipore Sigma | Cat#AB144P; RRID:AB_2079751; Lot 3675895 | (1:500) |
| Antibody | Mouse monoclonal Anti-CGRP | Abcam | Cat#ab81887; RRID: AB_1658411; Lots GR3336715-4, GR3374844-3, GR3233139-4 | (1:500–1:1,000) |
| Antibody | Chicken polyclonal Anti-GFP | Aves | Cat#GFP-1020; RRID: AB_10000240; Lot GFP3717982 | (1:2,000) |
| Antibody | Rabbit monoclonal Anti-NPY | Cell Signaling | Cat#11976; RRID: AB_2716286; Lots 3, 4, 5 | (Brainstem: 1:500) (Cochlea: 1:1,000) |
| Antibody | Guinea Pig polyclonal Anti-Parvalbumin | Synaptic Systems | Cat#195–004; RRID: AB_2156476; Lots 3–33, 3–36 | (1:1,000) |
| Antibody | Mouse monoclonal Anti-Synaptophysin 1 | Synaptic Systems | Cat#101 011; RRID: AB_887824; Lot 1–58 | (1:600) |
| Antibody | Chicken polyclonal Anti-TH | Abcam | Cat#ab76442; RRID: AB_1524535; Lot GR3393939-1 | (1:1,000) |
| Antibody | Rabbit polyclonal Anti-Ucn Polyclonal | Sigma-Alridch | Cat#SAB4503058; RRID:AB_10753229; Lot 310386 | (1:500) |
| Antibody | Donkey polyclonal Anti-Chicken 488 | Jackson ImmunoResearch | Cat#703-545-155; RID:AB_2340375 | (1:1,000) |
| Antibody | Donkey polyclonal Anti-Chicken 647 | Jackson ImmunoResearch | Cat#703-605-155; RRID:AB_2340379 | (1:1,000) |
| Antibody | Donkey polyclonal Anti-Goat 405 | Jackson ImmunoResearch | Cat#705-475-003; RRID:AB_2340426 | (1:1,000) |
| Antibody | Donkey polyclonal Anti-Goat 488 | Jackson ImmunoResearch | Cat#705-545-147; RRID:AB_2336933 | (1:1,000) |
| Antibody | Donkey polyclonal Anti-Goat 647 | ThermoFisher | Cat#A21447; RRID: AB_2535864 | (1:1,000) |
| Antibody | Donkey polyclonal Anti-Mouse 488 | Abcam | Cat#ab150105; RRID: AB_2732856 | (1:1,000) |
| Antibody | Donkey polyclonal Anti-Mouse 488 | Invitrogen | Cat#A21202; RRID: AB_141607 | (1:1,000) |
| Antibody | Donkey polyclonal Anti-Mouse 568 | ThermoFisher | Cat#A10037; RRID: AB_2534013 | (1:1,000) |
| Antibody | Donkey polyclonal Anti-Mouse 647 | ThermoFisher | Cat#A31571; RRID:AB_162542 | (1:1,000) |
| Antibody | Donkey polyclonal Anti-Rabbit 568 | ThermoFisher | Cat#A10042; RRID: AB_2534017 | (1:1,000) |
| Antibody | Donkey polyclonal Anti-Rabbit 647 | ThermoFisher | Cat#A31573; RRID:AB_2536183 | (1:1,000) |
| Antibody | Donkey polyclonal Anti-Guinea Pig 647 | Jackson ImmunoResearch | Cat#706–605–148; RRID:AB_2340476 | (1:1,000) |
| Sequence-based reagent | Cadps2 FISH probe | Molecular Instruments | Custom probe | |
| Sequence-based reagent | Calca FISH probe | Molecular Instruments | Calca | |
| Sequence-based reagent | Col4a4 FISH probe | Molecular Instruments | Custom probe | |
| Sequence-based reagent | Zfp804a FISH probe | Molecular Instruments | Custom probe | |
| Sequence-based reagent | Penk FISH probe | IDT | This paper | Sequence in Supplementary file 2 |
| Sequence-based reagent | Pdyn FISH probe | IDT | This paper | Sequence in Supplementary file 2 |
| Commercial assay or kit | 3’ Single Cell Kit: 10x v2 | 10x Genomics | Cat#PN-120267 | |
| Commercial assay or kit | 3’ Single Cell Kit: 10x v3 | 10x Genomics | Cat#PN-1000092 | |
| Commercial assay or kit | HCR RNA-FISH (Tissue section kit) | Molecular Instruments | N/A | |
| Commercial assay or kit | SPRIselect Reagent Kit | Beckman Coulter | Cat#B23318 | |
| Commercial assay or kit | DynaBeads MyOne Silane Beads | ThermoFisher | Cat#37002D | |
| Chemical compound, drug | 10% Tween | Bio-Rad | Cat#1610781 | |
| Chemical compound, drug | 10x PBS | ThermoFisher | Cat#70011044 | |
| Chemical compound, drug | 16% Paraformaldehyde | Electron Microscopy Sciences | Cat#15710 | |
| Chemical compound, drug | 1x PBS | ThermoFisher | Cat#10010031 | |
| Chemical compound, drug | 50% Glycerol | Ricca Chemical Company | Cat#3290–32 | |
| Chemical compound, drug | AlexaFluor-488 hydrazide | Fisher Scientific | Cat#A10436 | |
| Chemical compound, drug | APV | Tocris | Cat#0106 | |
| Chemical compound, drug | Biocytin | Sigma-Aldrich | Cat#B4261–25MG | |
| Chemical compound, drug | BSA | Sigma-Aldrich | Cat#A2058–5G | |
| Chemical compound, drug | BSA | ThermoFisher | Cat#BP1600–100 | |
| Chemical compound, drug | Buffer EB | Qiagen | Cat#19086 | |
| Chemical compound, drug | CaCl2 | Sigma-Aldrich | Cat#C7902 | |
| Chemical compound, drug | Corn Oil | Sigma-Aldrich | Cat#C8267–500ml | |
| Chemical compound, drug | CsCl | Fisher Scientific | Cat#AC206320250 | |
| Chemical compound, drug | CsOH | Fisher Scientific | Cat#AC213601000 | |
| Chemical compound, drug | D-gluconic acid | Sigma-Aldrich | Cat#P1847–-100G | |
| Chemical compound, drug | DEPC (Diethyl Pyrocarbonate) | Sigma-Aldrich | Cat#D5758 | |
| Chemical compound, drug | Dextrose | Fisher Scientific | Cat#D16-500 | |
| Chemical compound, drug | DNQX | Sigma-Aldrich | Cat#D0540 | |
| Chemical compound, drug | DTT | Sigma-Aldrich | Cat#D0632 | |
| Chemical compound, drug | EGTA | Fisher Scientific | Cat#409910250 | |
| Chemical compound, drug | Ethanol | Decon Labs | Cat#V1016 | |
| Chemical compound, drug | Fluoromount-G | Southern Biotech | Cat#0100–01 | |
| Chemical compound, drug | Formalin | ThermoFisher | Cat#F79-500 | |
| Chemical compound, drug | Gelatin | Sigma-Aldrich | Cat#G1890–100G | |
| Chemical compound, drug | Glutaraldehyde | ThermoFisher | Cat#50–262–19 | |
| Chemical compound, drug | HEPES | Sigma-Aldrich | Cat#H3375 | |
| Chemical compound, drug | IGEPAL CA-630 | Sigma-Aldrich | Cat#I8896 | |
| Chemical compound, drug | Iodixanol, OptiPrep Density Medium | Sigma-Aldrich | Cat#D1556 | |
| Chemical compound, drug | KCl | Fisher Scientific | Cat#P217–500 | |
| Chemical compound, drug | KCl | Sigma-Aldrich | Cat#P9541–500G | |
| Chemical compound, drug | KH2PO4 | Fisher Scientific | Cat#P288-100 | |
| Chemical compound, drug | KH2PO4 | Sigma-Aldrich | Cat#P9791–500G | |
| Chemical compound, drug | KOH | Sigma-Aldrich | Cat#P4494-50ML | |
| Chemical compound, drug | Kynurenic acid | Sigma-Aldrich | Cat#K3375-5G | |
| Chemical compound, drug | Low TE Buffer | ThermoFisher | Cat#12090–015 | |
| Chemical compound, drug | Mg-ATP | Sigma-Aldrich | Cat#A9187 | |
| Chemical compound, drug | MgCl2 | Sigma-Aldrich | Cat#M2670 | |
| Chemical compound, drug | MgSO4 | Fisher Scientific | Cat#M65-500 | |
| Chemical compound, drug | Na-GTP | Sigma-Aldrich | Cat#G8877 | |
| Chemical compound, drug | Na2-phosphocreatine | Sigma-Aldrich | Cat#P7936-10MG | |
| Chemical compound, drug | Na2HPO4 | Sigma-Aldrich | Cat#S3264-1KG | |
| Chemical compound, drug | NaCl | Fisher Scientific | Cat#S271–1 | |
| Chemical compound, drug | NaCl | Sigma-Aldrich | Cat#S3014-1KG | |
| Chemical compound, drug | NaHCO3 | Sigma-Aldrich | Cat#S5761–500G | |
| Chemical compound, drug | NEG-50 | Epredia | Cat#6502 | |
| Chemical compound, drug | Normal Donkey Serum | Jackson ImmunoResearch | Cat#017–000–121 | |
| Chemical compound, drug | Nuclease-Free Water | ThermoFisher | Cat#AM9937 | |
| Chemical compound, drug | Protease inhibitor cocktail | Roche | Cat#11836170001 | |
| Chemical compound, drug | Proteinase K | Sigma-Aldrich | Cat#P-6556 | |
| Chemical compound, drug | QX-314 | Fisher Scientific | Cat#23–135–0 | |
| Chemical compound, drug | RNAsin Plus RNase Inhibitor | Promega | Cat#N2615 | |
| Chemical compound, drug | Sodium Citrate: C6H5Na3O7∙2H2O | Sigma-Aldrich | Cat#S4641–1KG | |
| Chemical compound, drug | Spermidine trihydrochloride | Sigma-Aldrich | Cat#S2501 | |
| Chemical compound, drug | Spermine tetrahydrochloride | Sigma-Aldrich | Cat#S1141 | |
| Chemical compound, drug | Sucrose | Sigma-Aldrich | Cat#S0389–500G | |
| Chemical compound, drug | Tamoxifen | Sigma-Aldrich | Cat#T5648–1G | |
| Chemical compound, drug | Tetrodotoxin (TTX) | Cayman Chemical | Cat#14964 | |
| Chemical compound, drug | Tricine | Sigma-Aldrich | Cat#T5816–100G | |
| Chemical compound, drug | Triton X-100 | Sigma-Aldrich | Cat#T9284 | |
| Chemical compound, drug | Tween 20 | Sigma-Aldrich | Cat#P9416–100ML | |
| Chemical compound, drug | β-estradiol | Sigma-Aldrich | Cat#E2758–1G | |
| Software, algorithm | MultiClamp 700B | Molecular devices | ||
| Software, algorithm | DigiData 1440 A | Molecular devices | ||
| Software, algorithm | Clampex 10.6 software | Molecular devices | ||
| Software, algorithm | Origin 2021 | OriginLabs | ||
| Software, algorithm | Adobe Illustrator 2021 | Adobe | ||
| Software, algorithm | Inkscape | inkscape.org | ||
| Software, algorithm | R | https://www.r-project.org/ | v.3.5.3–4.0.5 | |
| Software, algorithm | Seurat | Stuart et al., 2019 | v.3.1.4–3.2.3 | |
| Software, algorithm | Imaris | Bitplane AG | v.9.8–9.9.0 | |
| Software, algorithm | MATLAB | MathWorks | R2020a, R2021b | |
| Software, algorithm | cellranger | 10x Genomics | v.2.1.0–3.0.2 | |
| Software, algorithm | CellPose | Stringer et al., 2021 | v.1 | |
| Software, algorithm | Prism | GraphPad | v.9.3.1 | |
| Software, algorithm | Zeiss ZEN | Zeiss | 2.3 (blue edition) | |
| Software, algorithm | Image processing: Local Normalization | Zhang et al., 2021 | https://github.com/xzhang03/Local-normalize | |
| Software, algorithm | ImageJ | National Institutes of Health | ||
| Software, algorithm | Measure_Line ImageJ Plugin | Eaton Peabody Labs, MEEI | https://meeeplfiles.partners.org/Measure_line.class | |
| Other | Brain matrix | Zivic Instruments | Cat#BSMNS001–1 | See Methods, “Single- nucleus sequencing” |
| Other | FlowMi cell strainer | Sigma | Cat#BAH136800040 | See Methods, “Single- nucleus sequencing” |
| Other | DAPI | Invitrogen | Cat#D1306 | DNA dye, (1:10,000) See Methods, “Single-nucleus sequencing” & “Immunofluorescence” |
| Other | Draq7 | Abcam | Cat#AB109202 | DNA dye, (1:100) See Methods, “Single-nucleus sequencing” |
Additional files
-
Supplementary file 1
Cell counts of NPY- and Ucn-expressing LOCs (related to Figure 3).
LOCs were manually annotated as expressing high or low levels of NPY or Ucn. Cells where both peptides were expressed above background are considered to be co-expressing both peptides. Each row includes cell counts from a single, 40 µm-thick section.
- https://cdn.elifesciences.org/articles/83855/elife-83855-supp1-v2.xlsx
-
Supplementary file 2
Custom FISH probe sequences (related to Figure 3, Figure 3—figure supplement 1).
Oligo probe sequences used in Figure 3—figure supplement 1 for detecting Penk and Pdyn via hybridization chain reaction-based FISH.
- https://cdn.elifesciences.org/articles/83855/elife-83855-supp2-v2.xlsx
-
Supplementary file 3
Physiological properties of LOCs do not correlate with Calca-GFP fluorescence intensity (related to Figure 6).
Results of single linear regression and correlation analysis between Calca-GFP and the indicated measures.
- https://cdn.elifesciences.org/articles/83855/elife-83855-supp3-v2.docx
-
Supplementary file 4
Results of statistical tests comparing peptide expression between OCN groups at different ages (related to Figure 7).
Adjusted p-values from Kruskal-Wallis multiple-comparisons test for the indicated cell types and ages. Data is shown in Figure 7A–D.
- https://cdn.elifesciences.org/articles/83855/elife-83855-supp4-v2.docx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/83855/elife-83855-mdarchecklist1-v2.docx
